Encapsulation of enrofloxacin in liposomes I: preparation and in vitro characterization of LUV

Liposomes are effectively used in the treatment of microbial infections. Higher cellular uptake has been reported when antibiotics are encapsulated in liposomes. In this study, enrofloxacin (ENF) was encapsulated in large unilamellar vesicles (LUVs) and the effects of formulation variables on the liposome characteristics were investigated. Liposomes were prepared using dry lipid film method. A number of variables such as molar ratios of phospholipid (DPPC; DL-alpha-phosphatidylcholine dipalmitoyl), cholesterol, ENF and amount of alpha-tocopherol and the volumes of internal (chloroform) and external phases [phosphate buffered saline PBS (pH 7.4)] were studied. In vitro characterization of the liposomes including the encapsulation capacity, size and drug release properties were carried out. Using of this method, spherical LUV liposomes with high drug content could be produced. Particle size of liposomes changed between 3.12 and 4.95 microm. The molar ratios of DPPC, cholesterol and ENF affected the size of the liposome (p < 0.05). The drug encapsulation capacities were high and changed between 37.1% and 79.5%. The highest ENF encapsulation was obtained with the highest cholesterol content. An increase in the drug encapsulation capacity of the liposome was found with increasing molar ratios of DPPC, cholesterol and ENF (p < 0.05). Furthermore, the release of ENF from the liposomes decreased as the molar ratios of DPPC, cholesterol and ENF increased (p < 0.05). In conclusion, a convenient colloidal carrier for the controlled release of ENF can be prepared by changing the formulation parameters of LUVs.     

Aberrant CD40-Induced NF-κB Activation in Human Lupus B Lymphocytes

Auto-reactive B lymphocytes and its abnormal CD40 signaling play important roles in the pathogenesis of systemic lupus erythematosus (SLE). In this study, we analyzed CD40 expression and CD40/CD154 induced activation of NF-κB signaling pathway in B cells from SLE patients. B cells from healthy volunteers and tonsilar B cells from chronic tonsillitis were used as negative and positive controls. Results showed CD40-induced NF-κB signaling was constitutively activated in B cells from active lupus patients, including decreased CD40 in raft portion, increased phosphorylation and degradation of IκBα, phosphorylation of P65, as well as increased nuclear translocation of P65, P50, c-Rel, which could be blocked by anti-CD154. CD154 stimulation could induce further phosphorylation and degradation of IκBα, as well as phosphorylation of P65 and nuclear translocation of P65. In addition, CD40-induced kinase activities in B cells from lupus patients mimicked that of tonsil B cells, in that IKKα/β were more activated compared to normal B cells. CD40-induced NF-κB activity was blocked by both IκB phosphorylation and proteosome degradation inhibitors in both lupus and normal B cells. All together, our findings revealed that canonical NF-κB signaling is constitutively activated in active lupus and is mediated by CD154/CD40. CD40 induced NF-κB activation is different in human lupus B lymphocytes compared with normal B cells.     

Ezrin-radixin-moesin-binding sequence of PSGL-1 glycoprotein regulates leukocyte rolling on selectins and activation of extracellular signal-regulated kinases

P-selectin glycoprotein ligand-1 (PSGL-1) mediates the capture (tethering) of free-flowing leukocytes and subsequent rolling on selectins. PSGL-1 interactions with endothelial selectins activate Src kinases and spleen tyrosine kinase (Syk), leading to α(L)β(2) integrin-dependent leukocyte slow rolling, which promotes leukocyte recruitment into tissues. In addition, but through a distinct pathway, PSGL-1 engagement activates ERK. Because ezrin, radixin and moesin proteins (ERMs) link PSGL-1 to actin cytoskeleton and because they serve as adaptor molecules between PSGL-1 and Syk, we examined the role of PSGL-1 ERM-binding sequence (EBS) on cell capture, rolling, and signaling through Syk and MAPK pathways. We carried out mutational analysis and observed that deletion of EBS severely reduced 32D leukocyte tethering and rolling on L-, P-, and E-selectin and slightly increased rolling velocity. Alanine substitution of Arg-337 and Lys-338 showed that these residues play a key role in supporting leukocyte tethering and rolling on selectins. Importantly, EBS deletion or Arg-337 and Lys-338 mutations abrogated PSGL-1-induced ERK activation, whereas they did not prevent Syk phosphorylation or E-selectin-induced leukocyte slow rolling. These studies demonstrate that PSGL-1 EBS plays a critical role in recruiting leukocytes on selectins and in activating the MAPK pathway, whereas it is dispensable to phosphorylate Syk and to lead to α(L)β(2)-dependent leukocyte slow rolling.     

Relative abundance and microhabitat use by the frog Geobatrachus walkeri (Anura: Strabomantidae) in two habitats of Sierra Nevada de Santa Marta, Colombia

Geobatrachus walkeri belongs to a monotypic frog genus endemic to the San Lorenzo area, Sierra Nevada de Santa Marta, Colombia. This species has been categorized as endangered because of its small distribution area and the decline in the extent and quality of its habitat. It inhabits two forest types with different composition and structure, the native secondary forest and a pine plantation (dominated by Pinus patula). To compare the relative abundance and microhabitat use of this species in these habitat types, 30 quadrants/environment were distributed randomly. The individual number, microhabitat use and other aspects of its natural history were registered using visual encounter surveys in both sites, including non-sampled areas in the quadrants. The relative abundance of frogs was significantly different between habitats and among seasons. The highest abundance of G. walkeri relative to the total area was found in the pine plantation, being 2.3 times higher than in the natural forest. More frogs were significantly found during the rainy season; nevertheless, active individuals were also found during the dry season. Significant differences were found in the microhabitat use with respect to the forest type and season. The most frequently microhabitat used in the two forest types was the pine leaf-litter; besides, in the native forest, the microhabitat occupied more frequently presented medium and large size stones. Geobatrachus walkeri is a successful species in pine plantations, associated permanently to its leaf-litter environment where it seems to develop its entire life cycle. The clear modifications in the soils and water, derived from the introduction of the pine plantation in this area, seem not to have negatively affected the conservation and successful maintenance of this species.     

Long-term exposure to a continuous 900 MHz electromagnetic field disrupts cerebellar morphology in young adult male rats

The pathological effects of exposure to an electromagnetic field (EMF) during childhood and adolescence may be greater than those from exposure during adulthood. We investigated possible pathological changes in the cerebellum of adolescent rats exposed to 900 MHz EMF daily for 25 days. We used three groups of six 21-day-old male rats as follows: unexposed control group (Non-EG), sham-exposed group (Sham-EG) and an EMF-exposed group (EMF-EG). EMF-EG rats were exposed to EMF in an EMF cage for 1 h daily from postnatal days 21 through 46. Sham-EG rats were placed in the EMF cage for 1 h daily, but were not subjected to EMF. No procedures were performed on the Non-EG rats. The cerebellums of all animals were removed on postnatal day 47, sectioned and stained with cresyl violet for histopathological and stereological analyses. We found significantly fewer Purkinje cells in the EMF-EG group than in the Non-EG and Sham-EG groups. Histopathological evaluation revealed alteration of normal Purkinje cell arrangement and pathological changes including intense staining of neuron cytoplasm in the EMF-EG group. We found that exposure to continuous 900 MHz EMF for 1 h/day during adolescence can disrupt cerebellar morphology and reduce the number of Purkinje cells in adolescent rats.     

Antigen Specificity of �æ� T Cells Depends Primarily on the Flanking Sequences of CDR3��

The structural basis that determines the specificity of γδ T cell receptor (TCR) recognition remains undefined. Our previous data show that the complementary determining region of human TCRδ (CDR3δ) is critical to ligand binding. Here we used linear and configurational approaches to examine the roles of V, N-D-N, or J regions in CDR3δ-mediated antigen recognition. Surprisingly, we found that the binding activities of CDR3δ from different γδ TCRs to their target tissues and ligands depend on the conserved flanking sequences (V and J) but not as much on the D region of CDR3δ fragment. We further defined the key residues in the V and J regions of CDR3δ fragments, including the cysteine residue in the V fragment and the leucine residue in the J fragment that determine their ligand binding specificity. Our results demonstrate that TCRδ primarily uses conserved flanking regions to bind ligands. This finding may provide an explanation for the limited number of γδ TCR ligands that have as yet been identified.Extensive studies suggest that γδ T cells play important roles in host defense against microbial infections, monitoring of tumorigenesis, immunoregulation, and development of autoimmunity (1–3). However, little is known about the structural basis of antigenic recognition by γδ T cell receptor (TCR)³ because of the limited identified specific ligands for γδ TCR and the lack of structural information revealing how γδ TCR might interact with such ligands.The crystallographic structure of a murine γδ TCR in complex with major histocompatibility complex class (MHC) Ib T22 (4, 5) showed that the CDR loops οf γδ TCR, predominantly germline-encoded residues of the complementary determining region of human TCRδ (CDR3δ), are in direct contact with T22, suggesting that the primary sequence of CDR3 in γδ TCR, especially CDR3δ, serves as a key determinant for the specificity of antigen recognition. Our recent finding that CDR3δ peptide mimics human γδ TCR binding to tumor cells and tissues is consistent with the role of CDR3δ in γδ TCR recognition (6).Based on this finding, we used synthesized CDR3δ peptide as a probe to screen putative protein ligands in tumor protein extracts by affinity chromatography analysis. With this novel strategy, we have successfully identified seven tumor-related epitopes, two hepatitis B virus (HBV) infection-related antigenic epitopes, and two self proteins including heat shock protein (HSP) 60 and human mutS homolog 2 (hMSH2) that are recognized by human γδ TCR (7). These results further support that the primary sequence of CDR3δ in γδ TCR determine the specificity of antigen binding.CDR3δ is composed of fragments derived from V, N-D-N, and J gene segments. The flanking sequences composed of V and J fragment is conserved while N-D-N region is diverse. The diversity of γδ TCRs is supposedly higher than that of TCRαβ due to the link of D gene fragment and the insertion of nucleotide acids (8). However, the number of identified antigenic ligands recognized by γδ TCR remains very limited. It has been demonstrated that γδ TCR recognizes some protein antigens and small phosphate or amine-containing compounds, including nonclassical MHC class I molecule T22 and T10 in mice (9), UL-16-binding protein (ULBP) (10) and mitochondrial F1-ATPase in humans (11). Nevertheless, important questions regarding γδ TCR recognition remain to be addressed. For example, given the seemingly high diversity of γδ TCR, why have only limited antigenic ligands been identified? What are the contributions of individual fragments of CDR3δ to antigen recognition? In αβ TCR, a single mutation in D gene fragment (12) abolishes its antigenic recognition, whereas the contribution of the different fragments in γδ TCR recognition remain unknown. Answers to these questions will shed important insights to antigen recognition of γδ T cells.In this study, we investigated the contribution of individual fragments of CDR3δ in antigen recognition. We mutated V, N-D-N, or J fragments of a Vδ2 TCR CDR3 sequence (OT3) in peptide and engineered γδ TCR. We found that the conserved flanking regions of CDR3δ play a critical role in antigenic binding to OEC cells/tissues or hMSH2 protein, a new ligand for γδTCR we found recently (7). Furthermore, we have identified the cysteine residue in V fragment and the leucine residue in J fragment as critical residues in the binding activity of γδ TCR. These results demonstrate that TCRδ chain uses the conserved flanking regions to recognize their antigens, suggesting that ligands for γδ ΤCR may also be conserved and limited in number.     

New insights into the systematics of North Atlantic Gaidropsarus (Gadiformes,Gadidae): flagging synonymies and hidden diversity

Gaidropsarus Rafinesque, 1810 is a genus of marine fishes, commonly known as rocklings, comprising 14 living species and showing a high ecological diversity from the intertidal zone to the deep sea. The systematics of this group has been controversial due to a general lack of representative specimens and the conservative morphology exhibited. A multidisciplinary approach combining the analysis of meristic data and the DNA barcode standard was applied in a species delimitation approach. Individuals representing eight valid and three unnamed species were collected, morphologically identified and archived in several museum collections. Comparison of DNA sequences shows complex results, furthering the idea of the difficult identification of specimens based on traditional taxonomy. DNA barcoding supports synonymies, like G. biscayensis–G. macrophthalmus and G. guttatus–G. mediterraneus, agreeing with the extensive overlaps observed in the meristic variables analysed and suggesting a reduction in the number of species. Genetic distances showed pairs of closely related species like G. granti–G. vulgaris and G. argentatus–G. ensis, the latter being only distinguished by one main distinctive character. Four deep-water specimens, morphologically classified only to the genus level, constituted three independent taxa apart from the ones present in this study and with no barcode matches in the repository databases. They could represent new records for the North Atlantic or unknown species of this genus. The results obtained show that more studies will be necessary to solve the systematics of this branch of the Gadiformes.     

Two new species of the <Emphasis Type="Italic">Fusarium solani</Emphasis> species complex isolated from compost and hibiscus (<Emphasis Type="Italic">Hibiscus</Emphasis> sp.)

Two new species in the Fusarium solani species complex (FSSC) are described and introduced. The new taxa are represented by German isolates CBS 142481 and CBS 142480 collected from commercial yard waste compost and vascular tissue of a wilting branch of hibiscus, respectively. The phylogenetic relationships of the collected strains to one another and within the FSSC were evaluated based on DNA sequences of 6 gene loci. Due to the limited sequence data available for reference strains in GenBank, however, a multi-gene phylogenetic analysis included partial sequences for the internal transcribed spacer region and intervening 5.8S nrRNA gene (ITS), translation elongation factor 1-alpha (tef1) and the RNA polymerase II second largest subunit (rpb2). Morphological and molecular phylogenetic data independently showed that these strains are distinct populations of the FSSC, nested within Clade 3. Thus, we introduce Fusarium stercicola and Fusarium witzenhausenense as novel species in the complex. In addition, 19 plant species of 7 legume genera were evaluated for their potential to host the newly described taxa. Eighteen plant species were successfully colonized, with 6 and 9 of these being symptomatic hosts for F. stercicola and F. witzenhausenense, respectively. As plants of the family Fabaceae are very distant to the originally sourced material from which the new taxa were recovered, our results suggest that F. stercicola and F. witzenhausenense are not host-specific and are ecologically fit to sustain stable populations in variety of habitats.     

Immunologic suppression of carcinogenesis in spontaneously hypertensive rats (SHR) with T cell depression

A strain of spontaneously hypertensive rats (SHR) showed a selective depression of T cell functions brought about by aging. Conversely, this strain had a high NK cell activity as compared to other normal rat strains. This SHR strain was found to be much more sensitive to the carcinogenic activity of low doses of MCA than were WKA rats with normal T cell functions. Allogeneic thymus grafts almost completely restored the T cell functions of SHR, whereas injection of an immunopotentiator, NSP, enhanced NK cell activity and also caused a partial recovering of T cell functions. When immunologic restoration was achieved, generation of killer T cells to syngeneic SMT-5 tumor cells was induced and the cytotoxic activity of NK cells to K-562 cells was also enhanced. But the cytotoxic activity to the SMT-5 cells of NK cells and macrophages from the treated or untreated SHR was not detected. Allogeneic thymus grafts induced a significant transplantation resistance against a syngeneic SMT-5 tumor and injection of NSP enhanced only the survival days of the rats. Allogeneic thymus grafts also significantly suppressed the incidence of tumors induced by MCA, whereas the injection of NSP was not effective in the prevention of tumor development but was effective in prolongation of latency periods. These results support the hypothesis that immune surveillance mediated by T cells is an important mechanism for the control of tumor development.     

Molecular phylogenetic analysis of Tulipa (Liliaceae) based on noncoding plastid and nuclear DNA sequences with an emphasis on Turkey

We investigated the phylogenetic relationships in Tulipa in Turkey using DNA sequences from the plastid trnL‐trnF region and the internal transcribed spacer (ITS) of nuclear ribosomal DNA. We generated trnL‐trnF and nuclear ITS sequences for 11 Tulipa spp. from Turkey and compared the utility of trnL‐trnF and ITS sequences for phylogenetic analysis. Neighbor‐joining, Bayesian and maximum parsimony methods were implemented using the same matrices. Our study of Tulipa based on molecular data revealed congruent results with previous studies. Despite the relatively lower resolution of trnL‐trnF than that of ITS, both sequence matrices generated similar results. Three clades were clearly distinguished, corresponding to subgenera Tulipa, Eriostemones and Orithyia. It is not fully resolved whether Clusianae should be recognized as a separate section of subgenus Tulipa or a distinct subgenus. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 270–279.