Density Functional study of the photoactive yellow protein's chromophore

We have investigated the structural and electronic properties of p-coumaric acid, the chromophore of the photoactive yellow protein (PYP), by means of first-principles molecular dynamics based on density functional theory (DFT). We have studied the chromophore both in the vacuum and in an extended model which includes the nearest residues in the binding pocket of PYP, as derived from crystallographic data. We have characterized the ground state of the isolated chromophore in its protonated and deprotonated forms and computed the energy barrier involved in the trans to cis isomerization process around the carbon-carbon double bond. A comparison of the optimized structures of the chromophore in the vacuum and in the extended protein model, both in the trans (ground state of PYP in the dark) and cis (first light-activated intermediate) configuration, shows how the protein environment affects the chromophore in the first step of the photocycle. Our model gives an energy storage of 25 kcal/mol associated with the trans-to-cia photoisomerization. Finally, we have elucidated the nature of the electronic excitation relevant for the photochemistry of PYP by means of time-dependent DFT calculations.

Histamine H3 receptor-mediated inhibition of sympathetically evoked mydriasis in rats

This study was designed to determine if the histamine H3 receptor agonist R-alpha-methylhistamine would play a role in modulation of sympathetically evoked mydriasis in anesthetized rats, and if so, to ascertain the specific receptor subtype(s) involved. Reproducible frequency-response curves of pupillary dilation were generated by stimulation of the cervical preganglionic sympathetic nerve (1-32 Hz). Systemic administration of R-alpha-methylhistamine (0.3-3.0 mg kg(-1)) produced a dose-related inhibition of the evoked mydriasis. The greatest inhibition was seen at lower frequency levels, with about 43% depression observed at 2 Hz. The specific histamine H3 receptor antagonist, clobenpropit (3.0 mg kg(-1), i.v.), blocked the inhibitory effect of R-alpha-methylhistamine, whereas neither the histamine H2 receptor antagonist, cimetidine (5.0 mg kg(-1), i.v.), nor the histamine H1 receptor antagonist, chlorpheniramine (0.5 mg kg(-1), i.v.), was effective. The histamine H2 receptor agonist, dimaprit (10 mg kg(-1), i.v.), was also without effect on the evoked mydriasis. R-alpha-methylhistamine (3.0 mg kg(-1)) did not inhibit phenylephrine-induced mydriasis. These results support the conclusion that R-alpha-methylhistamine produces inhibition of sympathetically evoked mydriasis via histamine H3 receptor stimulation, presumably by an action on presynaptic histamine H3 receptors.

Patterning and switching of nano-size ferroelectric memory cells

A "top-down" approach using a-beam lithography and a "bottom-up" one using self-assembly methods were used to fabricate ferroelelectric Pb(Zr,Ti)O-3, SrBi2Ta2O9 and BaTiO3 nanostructures with lateral sizes in the range of 30 nm to 100 nm. Switching of single sub-100 nm cells was achieved and piezoelectric hysteresis loops were recorded using a scanning force microscope working in piezoresponse mode. The piezoelectricity and its hysteresis acquired for 100 nm PZT cells demonstrate that a further decrease in lateral size under 100 nm appears to be possible and that the size effects are not fundamentally limiting on increase density of non-volatile ferroelectric memories in the Gbit range.

Relationships between EEA1 binding partners and their role in endosome fusion

Homotypic fusion between early endosomes requires the phosphatidylinositol 3-phosphate (PI3P)-binding protein, Early Endosomal Autoantigen 1 (EEA1). We have investigated the role of other proteins that interact with EEA1 in the fusion of early endosomes derived from Baby Hamster Kidney (BHK) cells. We confirm a requirement for syntaxin 13, but additionally show that the assay is equally sensitive to reagents specifically targeted against syntaxin 6. Binding of EEA1 to immobilised GST-syntaxin 6 and 13 was directly compared; only syntaxin 6 formed a stable complex with EEA1. Early endosome fusion requires the release of intravesicular calcium, and calmodulin plays a vital role in the fusion pathway, as judged by sensitivity to antagonists. We demonstrate that both EEA1 and syntaxin 13 interact with calmodulin. In the case of EEA1, binding to calmodulin requires an IQ domain, which is adjacent to a C-terminal FYVE domain that specifically binds to PI3P. We have assessed the influence of protein binding partners on EEA1 interaction with PI3P and find that both calmodulin and rab5-GTP are antagonistic to PI3P binding, whilst syntaxins 6 and 13 have no effect. These studies reveal a complex network of interactions between the proteins required for endosome fusion.

A microbiological study of Papillon-Lefévre syndrome in two patients

AIM: To analyse the microflora of subgingival plaque from patients with Papillon-Lefévre syndrome (PLS), which is a very rare disease characterised by palmar-plantar hyperkeratosis with precocious periodontal destruction.

METHODS: Bacterial isolates were identified using a combination of commercial identification kits, traditional laboratory tests, and gas liquid chromatography. Some isolates were also subjected to partial 16S rDNA sequencing. Plaque samples were also assayed for the presence of Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans in a quantitative enzyme linked immunosorbent assay (ELISA) using monoclonal antibodies.

RESULTS: The culture results showed that most isolates were capnophilic and facultatively anaerobic species-mainly Capnocytophaga spp and Streptococcus spp. The latter included S. constellatus, S. oralis, and S. sanguis. Other facultative bacteria belonged to the genera gemella, kingella, leuconostoc, and stomatococcus. The aerobic bacteria isolated were species of neisseria and bacillus. Anaerobic species included Prevotella intermedia, P. melaninogenica, and P. nigrescens, as well as Peptostreptococcus spp. ELISA detected P gingivalis in one patient in all sites sampled, whereas A. actinomycetemcomitans was detected in only one site from the other patient. Prevotella intermedia was present in low numbers.

CONCLUSIONS: Patients with PLS have a very complex subgingival flora including recognised periodontal pathogens. However, no particular periodontopathogen is invariably associated with PLS.