Ligand binding to heparan sulfate proteoglycans induces their aggregation and distribution along actin cytoskeleton.

Cell surface heparan sulfate proteoglycans (HSPGs) participate in molecular events that regulate cell adhesion, migration, and proliferation. The present study demonstrates that soluble heparin-binding proteins or cross-linking antibodies induce the aggregation of cell surface HSPGs and their distribution along underlying actin filaments. Immunofluorescence and confocal microscopy and immunogold and electron microscopy indicate that, in the absence of ligands, HSPGs are irregularly distributed on the fibroblast cell surface, without any apparent codistribution with the actin cytoskeleton. In the presence of ligand (lipoprotein lipase) or antibodies against heparan sulfate, HSPGs aggregate and colocalize with the actin cytoskeleton. Triton X-100 extraction and immunoelectron microscopy have demonstrated that in this condition HSPGs were clustered and associated with the actin filaments. Crosslinking experiments that use biotinylated lipoprotein lipase have revealed three major proteoglycans as binding sites at the fibroblast cell surface. These cross-linked proteoglycans appeared in the Triton X-100 insoluble fraction. Platinum/carbon replicas of the fibroblast surface incubated either with lipoprotein lipase or antiheparan sulfate showed large aggregates of HSPGs regularly distributed along cytoplasmic fibers. Quantification of the spacing between HSPGs by confocal microscopy confirmed that the nonrandom distribution of HSPG aggregates along the actin cytoskeleton was induced by ligand binding. When cells were incubated either with lipoprotein lipase or antibodies against heparan sulfate, the distance between immunofluorescence spots was uniform. In contrast, the spacing between HSPGs on fixed cells not incubated with ligand was more variable. This highly organized spatial relationship between actin and proteoglycans suggests that cortical actin filaments could organize the molecular machinery involved in signal transduction and molecular movements on the cell surface that are triggered by heparin-binding proteins.     

Ehrlich cell plasma membrane redox system is modulated through signal transduction pathways involvingcGMP and Ca2+ as second messengers

Ehrlich cell plasma membrane ferricyanide reductase activity increased in the presence of mastoparan, a generic activator of G proteins, using either whole cells or isolated plasma membrane fractions. Agents that increase intracellularcAMP also increased the rate of ferricyanide reduction by Ehrlich cells. For the first time, evidence is shown on a modulation of plasma membrane redox system bycGMP. In fact, permeant analogs ofcGMP, dibutyrylcGMP, and 8-bromo-cGMP increased the rate of ferricyanide reduction by the Ehrlich cell plasma membrane redox system. Furthermore, specific inhibition ofcGMP-phosphodiesterases by dipyridamole was also accompanied by an enhancement in the rate of ferricyanide reduction. On the other hand, treatments expected to increase cytoplasmic Ca²⁺ concentrations were accompanied by a remarkable stimulation of the reductase activity. Taking all these data together, it seems that the Ehrlich cell plasma membrane redox system is under a multiple and complex regulation by different signal transduction pathways involving G proteins, cyclic nucleotides, and Ca²⁺ ions.     

Papillomavirus capsid binding and uptake by cells from different tissues and species.

The inability of papillomaviruses (PV) to replicate in tissue culture cells has hampered the study of the PV life cycle. We investigated virus-cell interactions by the following two methods: (i) using purified bovine PV virions or human PV type 11 (HPV type 11) virus-like particles (VLP) to test the binding to eukaryotic cells and (ii) using different VLP-reporter plasmid complexes of HPV6b, HPV11 L1 or HPV11 L1/L2, and HPV16 L1 or HPV16 L1/L2 to study uptake of particles into different cell lines. Our studies showed that PV capsids bind to a broad range of cells in culture in a dose-dependent manner. Binding of PV capsids to cells can be blocked by pretreating the cells with the protease trypsin. Penetration of PV into cells was monitored by using complexes in which the purified PV capsids were physically linked to DNA containing the gene for beta-galactosidase driven by the human cytomegalovirus promoter. Expression of beta-galactosidase occurred in < 1% of the cells, and the efficiency of PV receptor-mediated gene delivery was greatly enhanced (up to 10 to 20% positive cells) by the use of a replication-defective adenovirus which promotes endosomal lysis. The data generated by this approach further confirmed the results obtained from the binding assays, showing that PV enter a wide range of cells and that these cells have all functions required for the uptake of PV. Binding and uptake of PV particles can be blocked by PV-specific antisera, and different PV particles compete for particle uptake. Our results suggest that the PV receptor is a conserved cell surface molecule(s) used by different PV and that the tropism of infection by different PV is controlled by events downstream of the initial binding and uptake.     

Evidence for the involvement of ethylene in the expression of specific RNAs during maturation of the orange,a non-climacteric fruit

Twelve cDNAs corresponding to mRNAs inducible by ethylene were isolated by differential screening of a cDNA library from ethylene-treated Citrus sinensis fruits. Northern analysis of RNA extracted from flavedo of ethylene-treated fruits and from fruits at different maturation stages showed that some of the mRNAs corresponding to these cDNAs were regulated both by ethylene treatment and during fruit maturation. The effect of exogenous ethylene on leaves and of endogenous ethylene on flowers showed that gene induction was not restricted to the flavedo tissue. The possible role of ethylene during maturation of the non-climacteric Citrus fruit is discussed.     

The mite Varroa jacobsoni does not transmit American foulbrood from infected to healthy colonies

The present study was conducted to determine whether Varroa jacobsoni can transmit American foulbrood (AFB), caused by the bacterium Paenibacillus larvae to healthy colonies by the surface transport of spores. Five two-storey Langstroth colonies of Apis mellifera ligustica were infested by placing a sealed brood comb, with 10% Varroa prevalence, between the central brood combs of each colony. Two months later the colonies were inoculated with P. larvae by adding brood comb pieces with clinical signs of AFB (45±5 scales per colony). After 60 days the brood area was completely uncapped by means of dissecting needles and tweezers, separating the Varroa mites from the larvae and the collected mites were introduced at a rate of 51 per colony into four recipient hives placed in an isolated apiary. Twenty female Varroa specimens were separated at random and observed by SEM. Paenibacillus larvae spores were found on the dorsal shield surface and on idiosomal setae. All colonies died after 4–5 months due to a high incidence of varroosis. No clinical AFB symptoms or P. larvae spores were observed in microscopic preparations. It is concluded that Varroa jacobsoni does not transmit AFB from infected to healthy colonies; it does, however transport P. larvae spores on its surface.     

Airborne grass pollen in Granada (Spain)

We have carried out a study on the annual and daily pollen concentrations from Gramineae over four consecutive years in the atmosphere of Granada (Spain). Samples of pollen grains were collected by the volumetric method with the aid of a Burkard sporetrap. Gramineae, according both to their high sensitizing capacity and to data from allergologists, are responsible for many pollinoses diagnosed in this area. In this work, daily pollen levels from April to July are monitored and the variations identified are interpreted in relation to meteorological conditions. Results indicated that the highest airborne concentrations of Gramineae pollen were found in May and June, although the beginning and intensity of pollination have been variable during these 4 years.     

Plasmid rolling circle replication and its control

Abstract This review summarises current information on rolling circle replicating plasmids originally isolated from Gram-positive bacteria with a low guanine and cytosine content in their DNA. It focuses on the peculiar biological features of these small, high copy number plasmids that replicate via an asymmetric RC mechanism. The regulation of plasmid copy number is also discussed.     

First evidence of human meconium glycoasparagines

During a systematic study of carbohydrate material present inhuman meconium, in addition to the previously described mucins,glycolipids and free oligosaccharides, we have now characterizeda significant quantity of free glycoasparagines. These glycoasparagineshave been isolated from human meconium by a combination of ion-exchange,concanavalin A (ConA)-affinity and high-performance liquid (HPLC)chromatographies. Their structures have been established by400 MHz ¹H-NMR spectroscopy. These compounds are related toN-acetyllactosaminic type structures and are based on the commoncore These glycoasparagines are probably derived from both proteaseand partial exoglycosidase hydrolysis of fetal gastrointestinalN-glycosyl proteins. Their structures are discussed in the contextof the known catabolic pathways of N-glycans glycoasparagine N-glycosyl protein catabolism meconium NMR