The SWI/SNF chromatin-remodeling factor stimulates repair by human excision nuclease in the mononucleosome core particle

To investigate the role of chromatin remodeling in nucleotide excision repair, we prepared mononucleosomes with a 200-bp duplex containing an acetylaminofluorene-guanine (AAF-G) adduct at a single site. DNase I footprinting revealed a well-phased nucleosome structure with the AAF-G adduct near the center of twofold symmetry of the nucleosome core. This mononucleosome substrate was used to examine the effect of the SWI/SNF remodeling complex on the activity of human excision nuclease reconstituted from six purified excision repair factors. We found that the three repair factors implicated in damage recognition, RPA, XPA, and XPC, stimulate the remodeling activity of SWI/SNF, which in turn stimulates the removal of the AAF-G adduct from the nucleosome core by the excision nuclease. This is the first demonstration of the stimulation of nucleotide excision repair of a lesion in the nucleosome core by a chromatin-remodeling factor and contrasts with the ACF remodeling factor, which stimulates the removal of lesions from internucleosomal linker regions but not from the nucleosome core.     

Expression of protective antigen in transgenic plants: a step towards edible vaccine against anthrax

Protective antigen (PA) is the most potent molecule for vaccination against anthrax. In the present study, we have successfully integrated protective antigen gene in nuclear genome of tobacco plants by Agrobacterium mediated leaf-disc transformation method. Expression of protective antigen gene was detected by immunoblot analysis using antisera raised against purified PA. A distinct band of approximately 83kDa lighted up in the protein extracted from transformed plants while there was no such band in untransformed plants. The plant expressed PA showed biological activity just like native PA, which was demonstrated by cytolytic assay on macrophage like cell lines with lethal factor. This study establishes for the first time expression of PA gene in a plant system and thus marks the first milestone towards developing edible vaccine against anthrax.     

Chemoprevention of tumors: the role of RAR-beta

Chemoprevention can be defined as the use of specific natural or synthetic chemical agents to reverse, suppress, or prevent carcinogenic progression to invasive cancer. The knowledge of carcinogenic mechanisms provides the scientific rationale for chemoprevention. Epithelial carcinogenesis proceeds through multiple discernible stages of molecular and cellular alterations. Understanding of the multistep nature of carcinogenesis has evolved through highly controlled animal carcinogenesis studies, and these studies have identified three distinct phases: initiation, promotion and progression. Animal model studies have provided evidence that the development of cancer involves many different factors, including alterations in the structures and functions of different genes. Transitions between successive stages can be enhanced or inhibited in the laboratory by different types of agents, such activities providing the fundamental basis for chemoprevention.     

Involvement of the Rab27 binding protein Slac2c/MyRIP in insulin exocytosis

Rab27a is a GTPase associated with insulin-containing secretory granules of pancreatic beta-cells. Selective reduction of Rab27a expression by RNA interference did not alter granule distribution and basal secretion but impaired exocytosis triggered by insulin secretagogues. Screening for potential effectors of the GTPase revealed that the Rab27a-binding protein Slac2c/MyRIP is associated with secretory granules of beta-cells. Attenuation of Slac2c/MyRIP expression by RNA interference did not modify basal secretion but severely impaired hormone release in response to secretagogues. Although beta-cells express Myosin-Va, a potential partner of Slac2c/MyRIP, no functional link between the two proteins could be demonstrated. In fact, overexpression of the Myosin-Va binding domain of Slac2c/MyRIP did not affect granule localization and hormone exocytosis. In contrast, overexpression of the actin-binding domain of Slac2c/MyRIP led to a potent inhibition of exocytosis without detectable alteration in granule distribution. This effect was prevented by point mutations that abolish actin binding. Taken together our data suggest that Rab27a and Slac2c/MyRIP are part of a complex mediating the interaction of secretory granules with cortical actin cytoskeleton and participate to the regulation of the final steps of insulin exocytosis.     

Heparan sulfate proteoglycans mediate the invasion of cardiomyocytes by Trypanosoma cruzi

Cytoadherence is an important step for the invasion of a mammalian host cell by Trypanosoma cruzi. Cell surface macromolecules are implicated in the T. cruzi-cardiomyocyte recognition process. Therefore, we investigated the role of cell surface proteoglycans during this invasion process and analyzed their expression after the parasite infected the target cells. Treatment of trypomastigote forms of T. cruzi with soluble heparan sulfate resulted in a significant inhibition in successful invasion, while chondroitin sulfate had no effect. Removal of sulfated glycoconjugates from the cardiomyocyte surface using glycosaminoglycan (GAG) lyases demonstrated the specific binding of the parasites to heparan sulfate proteoglycans. Infection levels were reduced by 42% whenthe host cells were previously treated with heparitinase II. No changes were detected in the expression of GAGs infected cardiomyocytes even after 96 h of infection. Our data demonstrate that heparan sulfate proteoglycans, but not chondroitin sulfate, mediate both attachment and invasion of cardiomyocytes by T. cruzi.     

First finding of Dirofilaria repens in a natural population of Aedes albopictus

The invasive mosquito Aedes albopictus (Skuse) (Diptera: Culicidae) has become widespread in Italy during the past decade. Also Italy has foci of canine filariasis caused by Dirofilaria (Spirurida: Onchocercidae), due to subcutaneous D. repens Railliet & Henry as well as the dog heartworm D. immitis (Leidy) transmitted by various vector mosquitoes (Diptera: Culicidae). In 2002, at Fiumicino, west of Rome (Lazio Region), 17% of dogs were found to have D. repens microfilariae in peripheral blood. To evaluate the role of Ae. albopictus as a vector of Dirofilaria in this area, female mosquitoes were collected daily, June-October 2002, landing on dog or human bait in a rural house at Focene. Mosquitoes were maintained at 27 degrees C and 70% RH for 6 days, to allow development or purging of filaria larvae, then identified and frozen for subsequent molecular assay with filaria-specific ribosomal S2-S16 primers. To distinguish specimens harbouring infective L3 Dirofilaria larvae, DNA was extracted separately from the mosquito abdomen and head-thorax. Dirofilaria species were identified by sequencing, confirmed by polymerase chain reaction of positive specimens using primers specific for D. immitis and D. repens. Dirofilaria DNA was detected in 3/154 (2%) of Ae. albopictus females examined: D. repens DNA in head-thorax and abdomen of one collected 27th July; D. immitis in the abdomen of one collected 24th September; DNA of both D. immitis and D. repens in the head-thorax of one collected 11th October 2002. Thus Ae. albopictus is a potential vector of both Dirofilarias in Italy, representing risks for veterinary and human health.     

Central neural mechanisms mediating human visceral hypersensitivity

Although visceral hypersensitivity is thought to be important in generating symptoms in functional gastrointestinal disorders, the neural mechanisms involved are poorly understood. We recently showed that central sensitization (hyperexcitability of spinal cord sensory neurones) may play an important role. In this study, we demonstrate that after a 30-min infusion of 0.15 M HCl acid into the healthy human distal esophagus, we see a reduction in the pain threshold to electrical stimulation of the non-acid-exposed proximal esophagus (9.6 +/- 2.4 mA) and a concurrent reduction in the latency of the N1 and P2 components of the esophageal evoked potentials (EEP) from this region (10.4 +/- 2.3 and 15.8 +/- 5.3 ms, respectively). This reduced EEP latency indicates a central increase in afferent pathway velocity and therefore suggests that hyperexcitability within the central visceral pain pathway contributes to the hypersensitivity within the proximal, non-acid-exposed esophagus (secondary hyperalgesia/allodynia). These findings provide the first electrophysiological evidence that central sensitization contributes to human visceral hypersensitivity.     

A cortical evoked potential study of afferents mediating human esophageal sensation

The aim of this study was to compare the characteristics of esophageal cortical evoked potentials (CEP) following electrical and mechanical stimulation in healthy subjects to evaluate the afferents involved in mediating esophageal sensation. Similarities in morphology and interpeak latencies of the CEP to electrical and mechanical stimulation suggest that they are mediated via similar pathways. Conduction velocity of CEP to either electrical or mechanical stimulation was 7.9-8.6 m/s, suggesting mediation via thinly myelinated Adelta-fibers. Amplitudes of CEP components to mechanical stimulation were significantly smaller than to electrical stimulation at the same levels of perception, implying that electrical stimulation activates a larger number of afferents. The latency delay of approximately 50 ms for each mechanical CEP component compared with the corresponding electrical CEP component is consistent with the time delay for the mechanical stimulus to distend the esophageal wall sufficiently to trigger the afferent volley. In conclusion, because the mechanical and electrical stimulation intensities needed to obtain esophageal CEP are similar and clearly perceived, it is likely that both spinal and vagal pathways mediate esophageal CEP. Esophageal CEP to both modalities of stimulation are mediated by myelinated Adelta-fibers and produce equally robust CEP responses. Both techniques may have important roles in the assessment of esophageal sensory processing in health and disease.     

Conformational analysis and stability of collagen peptides by CD and by 1H- and 13C-NMR spectroscopies

Four small type I collagen CNBr peptides containing complete natural sequences were purified from bovine skin and investigated by CD and 1H- and 13C-nmr spectroscopies to obtain information concerning their conformation and thermal stability. CD showed that a triple helix was formed at 10 degrees C in acidic aqueous solution by peptide alpha l(I) CB2 only, and to lesser extent, by alpha 1(I) CB4, whereas peptides alpha 1(I) CB5 and alpha 2(I) CB2 remained unstructured. Analytical gel filtration confirmed that peptides alpha 1(I) CB2 and alpha 1(I) CB4 only were able to form trimeric species at temperature between 14 and 20 degrees C, and indicated that the monomer = trimer equilibrium was influenced by the chaotropic nature of the salt present in the eluent, by its concentration, and by temperature variations. CD measurements at increasing temperatures showed that alpha 1(I) CB2 was less stable than its synthetic counterpart due to incomplete prolyl hydroxylation of the preparation from the natural source. 1H- and 13C-nmr spectra acquired in the temperature range 0-47 and 0-27 degrees C, respectively, indicated that with decreasing temperature the most abundant from of alpha 1(I) CB2 was in slow exchange with an assembled form, characterized by broad lines, as expected for the triple-helical conformation. A large number of trimer cross peaks was observed both in the proton and carbon spectra, and these were most likely due to the nonequivalence of the environments of the three chains in the triple helix. This nonequivalence may have implications for the aggregation of collagen molecules and for collagen binding to other molecules. The thermal transition from trimer to monomer was also monitored by 1H-nmr following the change in area of the signal belonging to one of the two beta protons of the C-terminal homoserine. The unfolding process was found to be fully reversible with a melting temperature of 13.4 degrees C, in agreement with CD results. The qualitative superposition of the melting curves obtained by CD for the peptide bond characteristics and by nmr for a side chain suggests that triple-helical backbone and side chains constitute a single unit.