Sialyllactose in viral membrane gangliosides is a novel molecular recognition pattern for mature dendritic cell capture of HIV-1

HIV-1 is internalized into mature dendritic cells (mDCs) via an as yet undefined mechanism with subsequent transfer of stored, infectious virus to CD4+ T lymphocytes. Thus, HIV-1 subverts a DC antigen capture mechanism to promote viral spread. Here, we show that gangliosides in the HIV-1 membrane are the key molecules for mDC uptake. HIV-1 virus-like particles and liposomes mimicking the HIV-1 lipid composition were shown to use a common internalization pathway and the same trafficking route within mDCs. Hence, these results demonstrate that gangliosides can act as viral attachment factors, in addition to their well known function as cellular receptors for certain viruses. Furthermore, the sialyllactose molecule present in specific gangliosides was identified as the determinant moiety for mDC HIV-1 uptake. Thus, sialyllactose represents a novel molecular recognition pattern for mDC capture, and may be crucial both for antigen presentation leading to immunity against pathogens and for succumbing to subversion by HIV-1.     

Biotransformation of indole derivatives by mycelial cultures

Biotransformation of tryptophan to tryptamine and 3-methyl-indole by Psilocybe coprophila was performed. On the other hand, Aspergillus niger was able to transform tryptophan to 5-hydroxy-tryptophan. P. coprophila biotransformed 5-hydroxy-tryptophan to 5-hydroxytryptamine. These results prove once more that fungi are good tools to establish hydroxyindole derivatives.     

Houseflies (Musca domestica) as Vectors for Extended-Spectrum β-Lactamase-Producing Escherichia coli on Spanish Broiler Farms

Flies may act as potential vectors for the spread of resistant bacteria to different environments. This study was intended to evaluate the presence of Escherichia coli strains resistant to cephalosporins in flies captured in the areas surrounding five broiler farms. Phenotypic and molecular characterization of the resistant population was performed by different methods: MIC determination, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and phylotyping. The presence of extended-spectrum beta-lactamase (ESBL) genes, their plasmid location, and the mobile genetic elements involved in their mobilization were studied. Additionally, the presence of 35 genes associated with virulence was evaluated. Out of 682 flies captured, 42 yielded ESBL-producing E. coli. Of these isolates, 23 contained bla_CTX-M-1, 18 contained bla_CTX-M-14, and 1 contained bla_CTX-M-9. ESBL genes were associated mainly with the presence of the IncI1 and IncFIB replicons. Additionally, all the strains were multiresistant, and five of them also harbored qnrS. Identical PFGE profiles were found for E. coli isolates obtained from flies at different sampling times, indicating a persistence of the same clones in the farm environment over months. According to their virulence genes, 81% of the isolates were considered avian-pathogenic E. coli (APEC) and 29% were considered extraintestinal pathogenic E. coli (ExPEC). The entrance of flies into broiler houses constitutes a considerable risk for colonization of broilers with multidrug-resistant E. coli. ESBLs in flies reflect the contamination status of the farm environment. Additionally, this study demonstrates the potential contribution of flies to the dissemination of virulence and resistance genes into different ecological niches.     

Novel series of substituted biphenylmethyl urea derivatives as MCH-R1 antagonists for the treatment of obesity

We have designed and synthesized two novel series of MCH-R1 antagonists based on a substituted biphenylmethyl urea core. SAR was explored, suggesting that optimal binding with the receptor was achieved when the biphenylmethyl group and the linker were substituted on the same nitrogen of the urea moiety. Compound 1-(3'-cyano-4-biphenylmethyl)-3-(2-hydroxy-1,1-dimethylethyl)-1-{2-[1-(4-methylbenzyl)-4-piperidinyl]ethyl}urea 2t showed the best antagonist binding activity to the MCH-R1 with a 43 nM K(i).     

Dynamic Localization of the Swe1 Regulator Hsl7 During the Saccharomyces cerevisiae Cell Cycle

In Saccharomyces cerevisiae, entry into mitosis requires activation of the cyclin-dependent kinase Cdc28 in its cyclin B (Clb)-associated form. Clb-bound Cdc28 is susceptible to inhibitory tyrosine phosphorylation by Swe1 protein kinase. Swe1 is itself negatively regulated by Hsl1, a Nim1-related protein kinase, and by Hsl7, a presumptive protein-arginine methyltransferase. In vivo all three proteins localize to the bud neck in a septin-dependent manner, consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1 complexes constitutes a checkpoint that monitors septin assembly. We show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1 in the absence of any other yeast proteins. With the use of both the two-hybrid method and in vitro binding assays, we found that Hsl7 contains distinct binding sites for Hsl1 and Swe1. A differential interaction trap approach was used to isolate four single-site substitution mutations in Hsl7, which cluster within a discrete region of its N-terminal domain, that are specifically defective in binding Hsl1. When expressed in hsl7Delta cells, each of these Hsl7 point mutants is unable to localize at the bud neck and cannot mediate down-regulation of Swe1, but retains other functions of Hsl7, including oligomerization and association with Swe1. GFP-fusions of these Hsl1-binding defective Hsl7 proteins localize as a bright perinuclear dot, but never localize to the bud neck; likewise, in hsl1Delta cells, a GFP-fusion to wild-type Hsl7 or native Hsl7 localizes to this dot. Cell synchronization studies showed that, normally, Hsl7 localizes to the dot, but only in cells in the G1 phase of the cell cycle. Immunofluorescence analysis and immunoelectron microscopy established that the dot corresponds to the outer plaque of the spindle pole body (SPB). These data demonstrate that association between Hsl1 and Hsl7 at the bud neck is required to alleviate Swe1-imposed G2-M delay. Hsl7 localization at the SPB during G1 may play some additional role in fine-tuning the coordination between nuclear and cortical events before mitosis.     

Coenzyme Q10 deficiency in mitochondrial DNA depletion syndromes

We evaluated coenzyme Q₁₀ (CoQ) levels in patients studied under suspicion of mitochondrial DNA depletion syndromes (MDS) (n = 39). CoQ levels were quantified by HPLC, and the percentage of mtDNA depletion by quantitative real-time PCR. A high percentage of MDS patients presented with CoQ deficiency as compared to other mitochondrial patients (Mann–Whitney-U test: p = 0.001). Our findings suggest that MDS are frequently associated with CoQ deficiency, as a possible secondary consequence of disease pathophysiology. Assessment of muscle CoQ status seems advisable in MDS patients since the possibility of CoQ supplementation may then be considered as a candidate therapy.     

High yield of endoreduplication induced by ICRF-193: a topoisomerase II catalytic inhibitor

Previous studies have demonstrated that phenolic compounds, including genistein (4',5,7-trihydroxyisoflavone) and resveratrol (3,4',5-trihydroxystilbene), are able to protect against carcinogenesis in animal models. This study was undertaken to examine the ability of genistein and resveratrol to inhibit reactive oxygen species (ROS)-mediated strand breaks in phi X-174 plasmid DNA. H(2)O(2)/Cu(II) and hydroquinone/Cu(II) were used to cause oxidative DNA strand breaks in the plasmid DNA. We demonstrated that the presence of genistein at micromolar concentrations resulted in a marked inhibition of DNA strand breaks induced by either H(2)O(2)/Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H(2)O(2) suggest that genistein may directly scavenge the ROS that participate in the induction of DNA strand breaks. In contrast to the inhibitory effects of genistein, the presence of resveratrol at similar concentrations led to increased DNA strand breaks induced by H(2)O(2)/Cu(II). Further studies showed that in the presence of Cu(II), resveratrol, but not genistein was able to cause DNA strand breaks. Moreover, both Cu(II)/Cu(I) redox cycle and H(2)O(2) were shown to be critically involved in resveratrol/copper-mediated DNA strand breaks. The above results indicate that despite their similar in vivo anticarcinogenic effects, genistein and resveratrol appear to exert different effects on oxidative DNA damage in vitro.     

The Klebsiella pneumoniae wabG gene: role in biosynthesis of the core lipopolysaccharide and virulence

To determine the function of the wabG gene in the biosynthesis of the core lipopolysaccharide (LPS) of Klebsiella pneumoniae, we constructed wabG nonpolar mutants. Data obtained from the comparative chemical and structural analysis of LPS samples obtained from the wild type, the mutant strain, and the complemented mutant demonstrated that the wabG gene is involved in attachment to alpha-L-glycero-D-manno-heptopyranose II (L,D-HeppII) at the O-3 position of an alpha-D-galactopyranosyluronic acid (alpha-D-GalAp) residue. K. pneumoniae nonpolar wabG mutants were devoid of the cell-attached capsular polysaccharide but were still able to produce capsular polysaccharide. Similar results were obtained with K. pneumoniae nonpolar waaC and waaF mutants, which produce shorter LPS core molecules than do wabG mutants. Other outer core K. pneumoniae nonpolar mutants in the waa gene cluster were encapsulated. K. pneumoniae waaC, waaF, and wabG mutants were avirulent when tested in different animal models. Furthermore, these mutants were more sensitive to some hydrophobic compounds than the wild-type strains. All these characteristics were rescued by reintroduction of the waaC, waaF, and wabG genes from K. pneumoniae.     

A mutation in the latency-related gene of bovine herpesvirus 1 inhibits protein expression from open reading frame 2 and an adjacent reading frame during productive infection

The latency-related (LR) gene of bovine herpesvirus 1 (BHV-1) is abundantly expressed during latency. A mutant BHV-1 strain that contains three stop codons at the 5′ terminus of the LR gene (LR mutant) does not reactivate from latency. This study demonstrates that the LR mutant does not express open reading frame 2 or an adjacent reading frame that lacks an initiating ATG (reading frame C). Since the LR mutant and wild-type BHV-1 express similar levels of LR RNA, we conclude that LR protein expression plays an important role in regulating the latency reactivation cycle in cattle.