Spondylodiscitis and an aortic aneurysm due to Campylobacter coli

The aim of the current study was to evaluate the prevalence of stump infections after major amputations of the lower extremities. Patients rehospitalized in Hospital de Base of the Medicine School in São José do Rio Preto in the period from January 2005 to January 2007 due to stump infection after major amputations of lower extremities were evaluated in a retrospective study. All the patients underwent prophylactic antibiotic therapy at the time of the surgery. The Fisher exact test was utilized for statistical analysis with an alpha error of 5% (p-value < 0.05) being considered acceptable. A total of 231 patients were submitted to major amputations during this period and 17 (7.3%) were rehospitalized due to amputation stump infections of which 5 (29.4%) died within one month. The association between death due to stump infection and other causes of death during rehospitalizations was not significant (Fisher exact test: p < 0.1). However, death during rehospitalizations was significantly higher than in the initial hospitalization.     

Genetic Resistance to Scrapie Infection in Experimentally Challenged Goats

In goats, several field studies have identified coding mutations of the gene encoding the prion protein (I/M₁₄₂, N/D₁₄₆, S/D₁₄₆, R/Q₂₁₁, and Q/K₂₂₂) that are associated with a lower risk of developing classical scrapie. However, the data related to the levels of resistance to transmissible spongiform encephalopathies (TSEs) of these different PRNP gene mutations are still considered insufficient for developing large-scale genetic selection against scrapie in this species. In this study, we inoculated wild-type (WT) PRNP (I₁₄₂R₁₅₄R₂₁₁Q₂₂₂) goats and homozygous and/or heterozygous I/M₁₄₂, R/H₁₅₄, R/Q₂₁₁, and Q/K₂₂₂ goats with a goat natural scrapie isolate by either the oral or the intracerebral (i.c.) route. Our results indicate that the I/M₁₄₂ PRNP polymorphism does not provide substantial resistance to scrapie infection following intracerebral or oral inoculation. They also demonstrate that H₁₅₄, Q₂₁₁, and K₂₂₂ PRNP allele carriers are all resistant to scrapie infection following oral exposure. However, in comparison to WT animals, the H₁₅₄ and Q₂₁₁ allele carriers displayed only moderate increases in the incubation period following i.c. challenge. After i.c. challenge, heterozygous K₂₂₂ and a small proportion of homozygous K₂₂₂ goats also developed the disease, but with incubation periods that were 4 to 5 times longer than those in WT animals. These results support the contention that the K₂₂₂ goat prion protein variant provides a strong but not absolutely protective effect against classical scrapie.     

A dual role for diacylglycerol kinase generated phosphatidic acid in autoantibody-induced neutrophil exocytosis

Dysregulated release of neutrophil azurophilic granules causes increased tissue damage and amplified inflammation during autoimmune disease. Antineutrophil cytoplasmic antibodies (ANCAs) are implicated in the pathogenesis of small vessel vasculitis and promote adhesion and exocytosis in neutrophils. ANCAs activate specific signal transduction pathways in neutrophils that have the potential to be modulated therapeutically to prevent neutrophil activation by ANCAs. We have investigated a role for diacylglycerol kinase (DGK) and its downstream product phosphatidic acid (PA) in ANCA-induced neutrophil exocytosis. Neutrophils incubated with the DGK inhibitor R59022, before treatment with ANCAs, exhibited a reduced capacity to release their azurophilic granules, demonstrated by a component release assay and flow cytometry. PA restored azurophilic granule release in DGK-inhibited neutrophils. Confocal microscopy revealed that R59022 did not inhibit translocation of granules, indicating a role for DGK during the process of granule fusion at the plasma membrane. In investigating possible mechanisms by which PA promotes neutrophil exocytosis, we demonstrated that exocytosis can only be restored in R59022-treated cells through simultaneous modulation of membrane fusion and increasing cytosolic calcium. PA and its associated pathways may represent viable drug targets to reduce tissue injury associated with ANCA-associated vasculitic diseases and other neutrophilic inflammatory disorders.     

Proteomic analyses of intermediate filaments reveals cytokeratin8 is highly acetylated--implications for colorectal epithelial homeostasis.

Butyrate is a critical cancer-preventive element in the colon milieu whose mechanism of action is unclear, but appears to be mediated through inhibition of histone deacetylases (HDACs) and consequent alterations in global protein acetylation. Cytokeratins (CKs) have roles in cytoskeletal function as components of the intermediate filaments (IFs) and this involves CKs in the regulation of tissue homeostasis of high-turnover epithelia such as the colon. We used a 2-D gel/MS analysis to characterise the proteome of IFs, and a novel monitoring-initiated detection and sequencing (MIDAS) approach to identify acetylation sites on principal proteins. We report that CKs are highly acetylated in a colon cancer cell line, with five acetylation sites characterised on CK8 and a further one on CK18. Acetylation of CK8 is responsive to butyrate. HDAC5 is the deacetylase associated with IFs. These data indicate a novel action of butyrate as a cancer preventive agent. Acetylation of CK8 may be associated with IFs stabilisation and thereby provide a candidate mechanism for the appropriate retention or loss of epithelial cells from the flat mucosa.     

Cationic liposomal lipids: from gene carriers to cell signaling

Cationic lipids are positively charged amphiphilic molecules which, for most of them, form positively charged liposomes, sometimes in combination with a neutral helper lipid. Such liposomes are mainly used as efficient DNA, RNA or protein carriers for gene therapy or immunization trials. Over the past decade, significant progress has been made in the understanding of the cellular pathways and mechanisms involved in lipoplex-mediated gene transfection but the interaction of cationic lipids with cell components and the consequences of such an interaction on cell physiology remains poorly described. The data reported in the present review provide evidence that cationic lipids are not just carriers for molecular delivery into cells but do modify cellular pathways and stimulate immune or anti-inflammatory responses. Considering the wide number of cationic lipids currently available and the variety of cellular components that could be involved, it is likely that only a few cationic lipid-dependent functions have been identified so far.     

Quantitative analysis of the binding of simian virus 40 large T antigen to DNA

SV40 large T antigen (T-ag) is a multifunctional protein that successively binds to 5'-GAGGC-3' sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork, and interacts with host DNA replication factors to promote replication of the simian virus 40 genome. The transition of T-ag from a sequence-specific binding protein to a nonspecific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for nonspecific double- and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild-type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5'-GAGGC-3' sequence and of the residue downstream of it, which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with an approximately 10-fold higher affinity than to nonspecific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer.     

Synthesis and biophysical evaluation of minor-groove binding C-terminus modified pyrrole and imidazole triamide analogs of distamycin

Five polyamide derivatives with rationally modified C-terminus moieties were synthesized and their DNA binding specificity and affinity determined. A convergent approach was employed to synthesize polyamides containing an alkylaminopiperazine (4 and 5), a truncated piperazine (6), or an alkyldiamino-C-terminus moiety (7 and 8) with two specific objectives: to investigate the effects of number of potential cationic centers and steric bulk at the C-terminus. CD studies confirmed that compounds 4, 5, 7, and 8 bind in the minor groove of DNA. The alkylpiperazine containing compounds (4 and 5) showed only moderate binding to DNA with DeltaT(m) values of 2.8 and 8.3 degrees C with their cognate sequence, respectively. The alkyldiamino compounds (7 and 8) were more impressive producing a DeltaT(m) of >17 and >22 degrees C, respectively. Compound 6 (truncated piperazine) did not stabilize its cognate DNA sequence. Footprints were observed for all compounds (except compound 6) with their cognate DNA sequence using DNase I footprinting, with compound 7 producing a footprint of 0.1 microM at the expected 5'-ACGCGT-3' site. SPR analysis of compound 7 binding to 5'-ACGCGT-3', 5'-ACCGGT-3', and 5'-AAATTT-3' produced binding affinities of 2.2x10(6), 3.3x10(5), and 1x10(5)M(-1), respectively, indicating a preference for its cognate sequence of 5'-ACGCGT-3'. These results are in good agreement with the footprinting data. The results indicate that steric crowding at the C-terminus is important with respect to binding. However, the number of cationic centers within the molecule may also play a role. The alkyldiamino-containing compounds (7 and 8) warrant further investigation in the field of polyamide research.