Enhancing membrane disruption by targeting and multivalent presentation of antimicrobial peptides

In order to enhance the membrane disruption of antimicrobial peptides both targeting and multivalent presentation approaches were explored. The antimicrobial peptides anoplin and temporin L were conjugated via click chemistry to vancomycin and to di- and tetravalent dendrimers. The vancomycin unit led to enhanced membrane disruption of large unilamellar vesicles (LUVs) displaying the vancomycin target lipid II, but only for temporin L and not for anoplin. The multivalent presentation led to enhanced LUV membrane disruption in the case of anoplin but not for temporin L.     

Galactose recognition by the apicomplexan parasite Toxoplasma gondii

Toxosplasma gondii is the model parasite of the phylum Apicomplexa, which contains numerous obligate intracellular parasites of medical and veterinary importance, including Eimeria, Sarcocystis, Cryptosporidium, Cyclospora, and Plasmodium species. Members of this phylum actively enter host cells by a multistep process with the help of microneme protein (MIC) complexes that play important roles in motility, host cell attachment, moving junction formation, and invasion. T. gondii (Tg)MIC1-4-6 complex is the most extensively investigated microneme complex, which contributes to host cell recognition and attachment via the action of TgMIC1, a sialic acid-binding adhesin. Here, we report the structure of TgMIC4 and reveal its carbohydrate-binding specificity to a variety of galactose-containing carbohydrate ligands. The lectin is composed of six apple domains in which the fifth domain displays a potent galactose-binding activity, and which is cleaved from the complex during parasite invasion. We propose that galactose recognition by TgMIC4 may compromise host protection from galectin-mediated activation of the host immune system.     

Giant DNA virus mimivirus encodes pathway for biosynthesis of unusual sugar 4-amino-4,6-dideoxy-D-glucose (Viosamine)

Mimivirus is one the largest DNA virus identified so far, infecting several Acanthamoeba species. Analysis of its genome revealed the presence of a nine-gene cluster containing genes potentially involved in glycan formation. All of these genes are co-expressed at late stages of infection, suggesting their role in the formation of the long fibers covering the viral surface. Among them, we identified the L136 gene as a pyridoxal phosphate-dependent sugar aminotransferase. This enzyme was shown to catalyze the formation of UDP-4-amino-4,6-dideoxy-D-glucose (UDP-viosamine) from UDP-4-keto-6-deoxy-D-glucose, a key compound involved also in the biosynthesis of L-rhamnose. This finding further supports the hypothesis that Mimivirus encodes a glycosylation system that is completely independent of the amoebal host. Viosamine, together with rhamnose, (N-acetyl)glucosamine, and glucose, was found as a major component of the viral glycans. Most of the sugars were associated with the fibers, confirming a capsular-like nature of the viral surface. Phylogenetic analysis clearly indicated that L136 was not a recent acquisition from bacteria through horizontal gene transfer, but it was acquired very early during evolution. Implications for the origin of the glycosylation machinery in giant DNA virus are also discussed.     

Identification of a Novel Pathway of Transforming Growth Factor-β1 Regulation by Extracellular NAD+ in Mouse Macrophages: IN VITRO AND IN SILICO STUDIES

Extracellular β-nicotinamide adenine dinucleotide (NAD(+)) is anti-inflammatory. We hypothesized that NAD(+) would modulate the anti-inflammatory cytokine Transforming Growth Factor (TGF)-β1. Indeed, NAD(+) led to increases in both active and latent cell-associated TGF-β1 in RAW 264.7 mouse macrophages as well as in primary peritoneal macrophages isolated from both C3H/HeJ (TLR4-mutant) and C3H/HeOuJ (wild-type controls for C3H/HeJ) mice. NAD(+) acts partially via cyclic ADP-ribose (cADPR) and subsequent release of Ca(2+). Treatment of macrophages with the cADPR analog 3-deaza-cADPR or Ca(2+) ionophores recapitulated the effects of NAD(+) on TGF-β1, whereas the cADPR antagonist 8-Br-cADPR, Ca(2+) chelation, and antagonism of L-type Ca(2+) channels suppressed these effects. The time and dose effects of NAD(+) on TGF-β1 were complex and could be modeled both statistically and mathematically. Model-predicted levels of TGF-β1 protein and mRNA were largely confirmed experimentally but also suggested the presence of other mechanisms of regulation of TGF-β1 by NAD(+). Thus, in vitro and in silico evidence points to NAD(+) as a novel modulator of TGF-β1.     

WT1 CpG islands methylation in human lung cancer: A pilot study

Background

CpG island hypermethylation of gene promoters and regulatory regions is a well-known mechanism of epigenetic silencing of tumor suppressors and is directly linked to carcinogenesis. Wilm’s tumor gene (WT1) is a tumor suppressor protein involved in the regulation of human cell growth and differentiation and a modulator of oncogenic K Ras signaling in lung cancer. Changes in the pattern of methylation of the WT1 gene have not yet been studied in detail in human lung cancer. In this study we compared the methylation profile of WT1 gene in samples of neoplastic and non-neoplastic lung tissue taken from the same patients.

Methods

DNA was extracted from neoplastic and normal lung tissue obtained from 16 patients with non small cell lung cancer (NSCLC). The methylation status of 29 CpG islands in the 5′ region of WT1 was determined by pyrosequencing. Statistical analysis was carried out by T test and Mann Whitney test.

Results

The mean percentage of methylation, considering all CpG islands of WT1 in the neoplastic tissues of the 16 NSCLC patients, was 16.2 ± 3.4, whereas in the normal lung tissue from the same patients it was 5.6 ± 1.7 (p < 0.001). Adenocarcinomas presented higher methylation levels than squamous cell carcinomas (p < 0,001).

Conclusions

Methylation of WT1 gene is significantly increased in NSCLC. Both histotype and exposure to cigarette smoke heavily influence the pattern of CpG islands which undergo hypermethylation.     

Cooperative stabilization of microtubule dynamics by EB1 and CLIP-170 involves displacement of stably bound P(i) at microtubule ends

End binding protein 1 (EB1) and cytoplasmic linker protein of 170 kDa (CLIP-170) are two well-studied microtubule plus-end-tracking proteins (+TIPs) that target growing microtubule plus ends in the form of comet tails and regulate microtubule dynamics. However, the mechanism by which they regulate microtubule dynamics is not well understood. Using full-length EB1 and a minimal functional fragment of CLIP-170 (ClipCG12), we found that EB1 and CLIP-170 cooperatively regulate microtubule dynamic instability at concentrations below which neither protein is effective. By use of small-angle X-ray scattering and analytical ultracentrifugation, we found that ClipCG12 adopts a largely extended conformation with two noninteracting CAP-Gly domains and that it formed a complex in solution with EB1. Using a reconstituted steady-state mammalian microtubule system, we found that at a low concentration of 250 nM, neither EB1 nor ClipCG12 individually modulated plus-end dynamic instability. Higher concentrations (up to 2 μM) of the two proteins individually did modulate dynamic instability, perhaps by a combination of effects at the tips and along the microtubule lengths. However, when low concentrations (250 nM) of EB1 and ClipCG12 were present together, the mixture modulated dynamic instability considerably. Using a pulsing strategy with [γ(32)P]GTP, we further found that unlike EB1 or ClipCG12 alone, the EB1-ClipCG12 mixture partially depleted the microtubule ends of stably bound (32)P(i). Together, our results suggest that EB1 and ClipCG12 act cooperatively to regulate microtubule dynamics. They further indicate that stabilization of microtubule plus ends by the EB1-ClipCG12 mixture may involve modification of an aspect of the stabilizing cap.     

Anatomy,pollen, and chromosomes of <Emphasis Type="Italic">Adenoa</Emphasis> (Turneraceae), a monotypic genus endemic to Cuba

The monotypic genus Adenoa is endemic to Cuba. Its name alludes to the presence of minute glands on the petal margin, identified in the present study as lachrymiform colleters. Here we describe the morphological, anatomical, palynological, and chromosome features that characterize Adenoa cubensis. The indumentum of Adenoa consists only of stellate trichomes. Unlike many species of the new world genera Piriqueta and Turnera, Adenoa lacks glandular hairs and extrafloral nectaries. Adenoa, Piriqueta, and Turnera share the presence of standard, sessile, and lachrymiform colleters. The leaves of Adenoa have xeromorphic features, which include entire, revolute blade margins, an adaxial hypodermis, and stomata restricted to the abaxial surface. The chromosome number is 2n?=?14, which is probably the ancestral number of the family. Adenoa chromosomes are similar in size to those of Turnera, and are larger than those of Piriqueta. Using the available data, we discuss relationships among the new world genera of Turneraceae.     

Influence of ovariectomy on cardiac oxidative stress in a renovascular hypertension model

The aim of this study was to evaluate the potential influence of endogenous ovarian hormones on cardiac oxidative stress in renovascular hypertension. Female Wistar rats (N = 10 per group) were divided among 4 groups: (i) normotensive control; (ii) hypertensive control; (iii) normotensive ovariectomized; and (iv) hypertensive ovariectomized rats. To induce hypertension, 2-kidney 1-clip (2K1C) Goldblatt's method was followed. Blood pressure (BP) was enhanced (25%) in 2K1C and it was not further altered in hypertensive ovariectomized animals. Lipid peroxidation (measured by thiobarbituric acid reactive substances; TBARS) increased in heart homogenates after ovariectomy (253%) and was additionally augmented when associated with hypertension (by 28%). Superoxide dismutase and catalase activities were similar in both hypertensive groups. Hypertension enhanced glutathione peroxidase activity (75%), but the association with ovariectomy prevented this change. Total radical trapping antioxidant potential (TRAP) decreased in hypertensive rats (34%) and was recovered when associated with ovariectomy. However, this adaptation seems not to be sufficient to avoid the increased oxidative damage in ovariectomized hypertensive animals. These results suggest a protective role for physiological ovarian hormones in the cardiac oxidative stress induced by 2K1C hypertension.     

Carbohydrate- and lipid-enriched meals acutely disrupt glycemic homeostasis by inducing transient insulin resistance in rats

Chronic intake of high-carbohydrate or high-lipid diets is a well-known insulin resistance inducer. This study investigates the immediate effect (1-6 h) of a carbohydrate- or lipid-enriched meal on insulin sensitivity. Fasted rats were refed with standard, carbohydrate-enriched (C), or lipid-enriched (L) meal. Plasma insulin, glucose, and non-esterified fatty acids (NEFA) were measured at 1, 2, 4, and 6 h of refeeding. The glucose-insulin index showed that either carbohydrates or lipids decreased insulin sensitivity at 2 h of refeeding. At this time point, insulin tolerance tests (ITTs) and glucose tolerance tests (GTTs) detected insulin resistance in C rats, while GTT confirmed it in L rats. Reduced glycogen and phosphorylated AKT and GSK3 content revealed hepatic insulin resistance in C rats. Reduced glucose uptake in skeletal muscle subjected to the fatty acid concentration that mimics the high NEFA level of L rats suggests insulin resistance in these animals is mainly in muscle. In conclusion, carbohydrate- or lipid-enriched meals acutely disrupt glycemic homeostasis, inducing a transient insulin resistance, which seems to involve liver and skeletal muscle, respectively. Thus, the insulin resistance observed when those types of diets are chronically consumed may be an evolution of repeated episodes of this transient insulin resistance.     

Morphological, molecular and functional differences of adult bone marrow- and adipose-derived stem cells isolated from rats of different ages

Adult mesenchymal stem cells have self-renewal and multiple differentiation potentials, and play important roles in regenerative medicine. However, their use may be limited by senescence or age of the donor, leading to changes in stem cell functionality. We investigated morphological, molecular and functional differences between bone marrow-derived (MSC) and adipose-derived (ASC) stem cells isolated from neonatal, young and old rats compared to Schwann cells from the same animals. Immunocytochemistry, RT-PCR, proliferation assays, western blotting and transmission electron microscopy were used to investigate expression of senescence markers. Undifferentiated and differentiated ASC and MSC from animals of different ages expressed Notch-2 at similar levels; protein-38 and protein-53 were present in all groups of cells with a trend towards increased levels in cells from older animals compared to those from neonatal and young rats. Following co-culture with adult neuronal cells, dMSC and dASC from animals of all ages elicited robust neurite outgrowth. Mitotracker^® staining was consistent with ultrastructural changes seen in the mitochondria of cells from old rats, indicative of senescence. In conclusion, this study showed that although the cells from aged animals expressed markers of senescence, aged MSC and ASC differentiated into SC-like cells still retain potential to support axon regeneration.