Human papillomavirus type 6b virus-like particles are able to activate the Ras-MAP kinase pathway and induce cell proliferation

The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the alpha6 integrin. The alpha6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. sere we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is Lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in fell proliferation, suggesting involvement of the Ras-MAP kinase pathway, Indeed, VLP binding results in rapid phosphorylation of the beta4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein She to beta4, Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c-myc mRNA was observed at 60 min, These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells.

A longitudinal study of interleukin-1 gene polymorphisms and periodontal disease in a general adult population

Background: Cross-sectional studies have demonstrated that a specific polymorphism (allele 2 of both IL-1A +4845 and IL-1B +3954) in the IL-1 gene cluster has been associated with an increased susceptibility to severe periodontal disease and to an increased bleeding tendency during periodontal maintenance. The aim of the present study was to investigate the relationship between IL-1 genotype and periodontitis in a prospective longitudinal study in an adult population of essentially European heritage. Methods: From an ongoing study of the Oral Care Research Programme of The University of Queensland, 295 subjects consented to genotyping for IL-1 allele 2 polymorphisms. Probing depths and relative attachment levels were recorded at baseline, 6, 12, 24, 36, 48 and 60 months using the Florida probe. Periodontitis progression at a given site was defined as attachment loss greater than or equal to2 mm at any observation period during the 5 years of the study and the extent of disease progression determined by the number of sites showing attachment loss. Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Prevotella intermedia were detected using ELISA. Results: 38.9% of the subjects were positive for the composite IL-1 genotype. A relationship between the IL-1 positive genotype and increased mean probing pocket depth in non-smokers greater than 50 years of age was found. Further, IL-1 genotype positive smokers and genotype positive subjects with P. gingivalis in their plaque had an increase in the number of probing depths greater than or equal to3.5 mm, There was a consistent trend for IL-1 genotype positive subjects to experience attachment loss when compared with IL-1 genotype negative subjects. Conclusion: The results of this study have shown an interaction of the IL-1 positive genotype with age, smoking and P. gingivalis which suggests that IL-1 genotype is a contributory but non-essential risk factor for periodontal disease progression in this population.

Analysis of acousto-ultrasonic characteristics for contact-type transducers coupled to composite laminated plates

The acousto-ultrasonic (AU) input-output characteristics for contact-type transmitting and receiving transducers coupled to composite laminated plates are considered in this paper. Combining a multiple integral transform method, an ordinary discrete layer theory for the laminates and some simplifying assumptions for the electro-mechanical transduction behaviour of the transducers, an analytical solution is developed which can deal with all the wave processes involved in the AU measurement system, i.e, wave generation, wave propagation and wave reception. The spectral response of the normal contact pressure sensed by the receiving transducer due to an arbitrary input pulse excited by the transmitting transducer is obtained. To validate the new analytical-numerical spectral technique in the low-frequency regime, the results are compared with Mindlin plate theory solutions. Based on the analytical results, numerical calculations are carried out to investigate the influence of various external parameters such as frequency content of the input pulse, transmitter/receiver spacing and transducer aperture on the output of the measurement system. The results show that the presented analytical-numerical procedure is an effective tool for understanding the input-output characteristics of the AU technique for laminated plates. (C) 2001 Elsevier Science Ltd. All rights reserved.

Differential perturbation of neuronal and glial glutamate transport systems in retinal ischaemia

Glutamate is the major excitatory neurotransmitter in the retina and is removed from the extracellular space by an energy-dependent process involving neuronal and glial cell transporters. The radial glial Muller cells express the glutamate transporter, GLAST, and preferentially accumulate glutamate. However, during an ischaemic episode, extracellular glutamate concentrations may rise to excitotoxic levels. Is this catastrophic rise in extracellular glutamate due to a failure of GLAST? Using immunocytochemistry, we monitored the transport of the glutamate transporter substrate, D-aspartate, in the retina under normal and ischaemic conditions. Two models of compromised retinal perfusion were compared: (1) Anaesthetised rats had their carotid arteries occluded for 7 days to produce a chronic reduction in retinal blood flow. Retinal function was assessed by electroretinography. D-aspartate was injected into the eye for 45 min, Following euthanasia, the retina was processed for D-aspartate. GLAST and glutamate immunocytochemistry. Although reduced retinal perfusion suppresses the electroretinogram b-wave, neither retinal histology, GLAST expression, nor the ability of Muller cells to uptake D-aspartate is affected. As this insult does not appear to cause excitotoxic neuronal damage, these data suggest that GLAST function and glutamate clearance are maintained during periods of reduced retinal perfusion. (2) Occlusion of the central retinal artery for 60 min abolishes retinal perfusion, inducing histological damage and electroretinogram suppression. Although GLAST expression appears to be normal. its ability to transport D-aspartate into Muller cells is greatly reduced. Interestingly, D-aspartate is transported into neuronal cells, i.e. photoreceptors, bipolar and ganglion cells. This suggests that while GLAST is vitally important for the clearance of excess extracellular glutamate, its capability to sustain inward transport is particularly susceptible to an acute ischaemic attack. Manipulation of GLAST function could alleviate the degeneration and blindness that result from ischaemic retinal disease. (C) 2001 Elsevier Science Ltd, All rights reserved.

Gill pathology caused by infestations of adult and preadult Dissonus manteri Kabata (Copepoda : Dissonidae) on coral trout, Plectropomus leopardus (Lacepede), (Serranidae)

Adult and preadult Dissonus manteri attached to the gills of Plectropomus leopardus cause significant pathology in the form of large hyperplastic nodules on the afferent (leading), edges of gill filaments. Nodules result from the dual actions of parasite attachment and feeding. The host response is characterized by severe epithelial hyperplasia, supplemented by fibroplasia and inflammation. Parasites attach close to the gill arch near the base of filaments. They have little effect on gill vasculature as the maxillipeds penetrate the filament superficial to the efferent filament artery and do not interfere with the blood vessels of the secondary lamellae. Tissue proliferation is limited to the wide portion of filament 'edge' epithelium in the proximal third and also does not extend to the secondary lamellae. Nodules are most numerous towards the ends of hemibranchs and are generally absent from the central regions. Leading hemibranchs bear significantly more nodules than their trailing counterparts. Of the total number of nodules, 20.5% are located on the pseudobranchs. Distribution patterns are considered to be primarily the result of D. manteri avoiding strong water currents, although this cannot explain the difference between numbers on leading and trailing hemibranchs.

Prospective survey of tick paralysis in dogs

Objective To obtain information on tick paralysis in dogs, including the nature of disease, host signalment, tick-host relationship, treatment, disease progression and recovery, and preventive measures. Design A prospective survey of 577 dogs affected by tick paralysis was conducted during 1998. Forty-two veterinary clinics along the eastern coast of Australia were instructed to complete survey forms for the first 15 dogs that presented with tick paralysis during September to November. Results Five percent of dogs died from tick paralysis. Younger dogs were more likely to survive. Long coat length was associated with a greater tick burden but not greater tick size, whereas coat thickness had no bearing on either. Dogs with mild disease recovered more quickly from tick paralysis. Respiratory and gait scores reflected disease severity and were good prognostic indicators. The size of the tick did not reflect the severity of the clinical condition it induced in the host. No method of tick removal or in situ treatment improved recovery time or reduced mortality. However, the time spent in hospital was significantly less for dogs from which the live tick was manually removed. Inspiratory strider. evident in some dogs with tick paralysis, was not related to tick attachment on the neck. The use of acepromazine maleate or dexamethasone did not reduce recovery time or mortality. Increasing the dose of tick antitoxin serum (TAS) above 0.1 mL/kg had no effect on mortality or recovery time. Dogs with severe disease that received an additional dose of TAS were significantly less likely to survive. Subcutaneous use of TAS at the site of tick attachment was of no benefit in reducing mortality or time to initial clinical improvement. A registered preventative product had not been used on the majority of dogs. Clipping the coat to search for ticks did not reduce mortality. Conclusions Therapy needs to address cardiopulmonary dysfunction that may be due directly to the effect of tick toxin and not just respiratory compromise caused by progressive respiratory muscle failure.

The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers

Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled-coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi. membranes. By immunoelectron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)-p230(GRIP) fusion protein, we show binding specifically to a subset of membranes of the trans-Golgi network (TGN). Real-time imaging of live HeLa cells revealed that the GFP-p230(GRIP) was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP-p230(GRIP) transfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFP-p230(GRIP) binds to a specific population of vesicles distinct from those labelled for beta -COP or gamma -adaptin. The GFP-p230(GRIP) fusion protein is recruited to the same vesicle population as full-length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans-Golgi network tubulovesicular carriers.

Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins

Smooth muscle cells (SMC) exhibit a functional plasticity, modulating from the mature phenotype in which the primary function is contraction, to a less differentiated state with increased capacities for motility, protein synthesis, and proliferation. The present study determined, using Western analysis, double-label immunofluorescence and confocal microscopy, whether changes in phenotypic expression of rabbit aortic SMC in culture could be correlated with alterations in expression and distribution of structural proteins. Contractile state SMC (days 1 and 3 of primary culture) showed distinct sorting of proteins into subcellular domains, consistent with the theory that the SMC structural machinery is compartmentalised within the cell. Proteins specialised for contraction (alpha -SM actin, SM-MHC, and calponin) were highly expressed in these cells and concentrated in the upper central region of the cell. Vimentin was confined to the body of the cell, providing support for the contractile apparatus but not co-localising with it. In line with its role in cell attachment and motility, beta -NM actin was localised to the cell periphery and basal cortex. The dense body protein alpha -actinin was concentrated at the cell periphery, possibly stabilising both contractile and motile apparatus. Vinculin-containing focal adhesions were well developed, indicating the cells' strong adhesion to substrate. In synthetic state SMC (passages 2-3 of culture), there was decreased expression of contractile and adhesion (vinculin) proteins with a concomitant increase in cytoskeletal proteins (beta -non-muscle [NM] actin and vimentin). These quantitative changes in structural proteins were associated with dramatic chan-es in their distribution. The distinct compartmentalisation of structural proteins observed in contractile state SMC was no longer obvious, with proteins more evenly distributed throughout die cytoplasm to accommodate altered cell function. Thus, SMC phenotypic modulation involves not only quantitative changes in contractile and cytoskeletal proteins, but also reorganisation of these proteins. Since the cytoskeleton acts as a spatial regulator of intracellular signalling, reorganisation of the cytoskeleton may lead to realignment of signalling molecules, which, in turn, may mediate the changes in function associated with SMC phenotypic modulation. (C) 2001 Wiley-Liss, Inc.

Thiopalmitoylation of myelin proteolipid protein epitopes enhances immunogenicity and encephalitogenicity

Proteolipid protein (PLP) is the most abundant protein of CNS myelin, and is posttranslationally acylated by covalent attachment of long chain fatty acids to cysteine residues via a thioester linkage. Two of the acylation sites are within epitopes of PLP that are encephalitogenic in SJL/J mice (PLP104-117 and PLP139-151) and against which increased immune responses have been detected in some multiple sclerosis patients. It is known that attachment of certain types of lipid side chains to peptides can result in their enhanced immunogenicity. The aim of this study was to determine whether thioacylated PLP peptides, as occur in the native protein, are more immunogenic than their nonacylated counterparts, and whether thioacylation influences the development of autoreactivity and experimental autoimmune encephalomyelitis. The results show that in comparison with nonacylated peptides, thioacylated PLP lipopeptides can induce greater T cell and Ab responses to both the acylated and nonacylated peptides. They also enhanced the development and chronicity of experimental autoimmune encephalomyelitis. Synthetic peptides in which the fatty acid was attached via an amide linkage at the N terminus were not encephalitogenic, and they induced greater proportions of CD8(+) cells in initial in vitro stimulation. Therefore, the lability and the site of the linkage between the peptide and fatty acid may be important for induction of encephalitogenic CD4(+) T cells. These results suggest that immune responses induced by endogenous thioacylated lipopeptides may contribute to the immunopathogenesis of chronic experimental demyelinating diseases and multiple sclerosis.

Pre- and postsynaptic actions of ATP on neurotransmission in rat submandibular ganglia

The pre- and postsynaptic actions of exogenously applied ATP were investigated in intact and dissociated parasympathetic neurotics of rat submandibular ganglia. Nerve-evoked excitatory postsynaptic potentials (EPSPs) were not inhibited by the purinergic receptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2 ' ,4 ' -disulphonic acid (PPADS), or the desensitising agonist, alpha,beta -methylene ATP. In contrast. EPSPs were abolished by the nicotinic acetylcholine receptor antagonists, hexamethonium and mecamylamine. Focal application of ATP (100 muM) had no effect on membrane potential of the postsynaptic neurone or on the amplitude of spontaneous EPSPs. Taken together, these results suggest the absence of functional purinergic (P2) receptors on the postganglionic neurone in situ. In contrast, focally applied ATP (100 muM) reversibly inhibited nerve-evoked EPSPs. Similarly, bath application of the non-hydrolysable analogue of ATP, ATP gammaS, reversibly depressed EPSPs amplitude, The inhibitory effects of ATP and ATP gammaS on nerve-evoked transmitter release were antagonised by bath application of either PPADS or suramin, suggesting ATP activates a presynaptic P2 purinoceptor to inhibit acetylcholine release from preganglionic nerves in the submandibular ganglia. In acutely dissociated postganglionic neurotics from rat submandibular ganglia. focal application of ATP (100 LM) evoked an inward current and subsequent excitatory response and action potential firing, which was reversibly inhibited by PPADS (10 muM). The expression of P2X purinoceptors in wholemount and dissociated submandibular ganglion neurones was examined using polyclonal antibodies raised against the extracellular domain of six P2X purinoceptor subtypes (P2X(1-6)). In intact wholemount preparations, only the P2X(5) purinoceptor subtype was found to be expressed in the submandibular ganglion neurones and no P2X immunoreactivity was detected in the nerve fibres innervating the ganglion. Surprisingly, in dissociated submandibular ganglion neurones, high levels of P2X(2) and P2X(4) purinoceptors immunoreactivity were found on the cell surface. This increase in expression of P2X(2) and P2X(4) purinoceptors in dissociated submandibular neurones could explain the increased responsiveness of the neurotics to exogenous ATP. We conclude that disruption of ganglionic transmission in vivo by either nerve damage or synaptic blockade may up-regulate P2X expression or availability and alter neuronal excitability. (C) 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Syntaxin 7 complexes with mouse Vps10p tail interactor 1b, Syntaxin 6, vesicle-associated membrane protein (VAMP)8, and VAMP7 in B16 melanoma cells

Syntaxin 7 is a mammalian target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7, Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), alpha -synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMPS to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.

Relationship between endosomes and lysosomes

Delivery of endocytosed macromolecules to lysosomes occurs by means of direct fusion of late endosomes with lysosomes. This has been formally demonstrated in a cell-free content mixing assay using late endosomes and lysosomes from rat liver. There is evidence from electron microscopy Studies that the same process occurs in intact cells. The fusion process results in the formation of hybrid organelles from which lysosomes are reformed. The discovery of the hybrid organelle has opened up three areas of investigation: (i) the mechanism of direct fusion of late endosomes and lysosomes, (ii) the mechanism of re-formation of lysosomes from the hybrid organelle, and (iii) the function of the hybrid organelle. Fusion has analogies with homotypic vacuole fusion in yeast. It requires syntaxin 7 as part of the functional trans-SNARE [SNAP receptor, where SNAP is soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein] complex and the release of lumenal calcium to achieve membrane fusion. Reformation of lysosomes from the hybrid organelle occurs by a maturation process involving condensation of lumenal content and probably removal of some membrane proteins by vesicular traffic. Lysosomes may thus be regarded as a type of secretory granule, storing acid hydrolases in between fusion events with late endosomes. The hybrid organelle is predicted to function as a 'cell stomach', acting as a major site of hydrolysis of endocytosed macromolecules.

Total synthesis and absolute stereochemistry of plakortone D

The first total synthesis of plakortone D is described and thereby establishes the structure and absolute stereochemistry of the most biologically active member of the marine-derived plakortone family. The sterically congested bicyclic lactone core results from a Pd(II)-induced hydroxycyclization−carbonylation−lactonization sequence on an enediol whose chirality was installed by AD-technology. Attachment of the side chain, also constructed using AD-methodology, was achieved by using a modified Julia coupling. The described approach enables acquisition of other plakortones and analogues, in the correct (natural) stereochemical series.

Air target detection via bistatic radar based on LEOS communication signals

The paper discusses the bistatic radar parameters for the case when the transmitter is a satellite emitting communication signals. The model utilises signals from an Iridium-like low earth orbiting satellite system. The maximum detection range, when thermal noise-limited, is discussed at the theoretical level and these results are compared with experimentation. Satellite-radar signal levels and the power of ground reflections are evaluated.

GABA(A) receptor sites in the developing human foetus

GABA(A) receptor sites were characterised in cerebral cortex tissue samples from deceased neurologically normal infants who had come to autopsy during the third trimester of pregnancy. Pharmacological parameters were obtained from homogenate binding studies which utilised the 'central-type' benzodiazepine ligands [H-3]diazepam and [H-3]flunitrazepam, and from the GABA activation of [H-3]diazepam binding. It was found that the two radioligands behaved differently during development. The affinity of [H-3]flunitrazepam for its binding site did not vary significantly between preparations, whereas the [H-3]diazepam K-D showed marked regional and developmental variations: infant tissues showed a distinctly lower affinity than adults for this ligand. The density of [H-3]flunitrazepam binding sites increased similar to35% during the third trimester to reach adult levels by term, whereas [H-3]diazepam binding capacity declined slightly but steadily throughout development. The GABA activation of [H-3]diazepam binding was less efficient early in the trimester, in that the affinity of the agonist was significantly lower, though it rose to adult levels by term. The strength of the enhancement response increased to adult levels over the same time-frame. The results strongly suggest that the subunit composition of cortical GABA(A) sites changes significantly during this important developmental stage. (C) 2002 Elsevier Science B.V. All rights reserved.