Fitness and Phenotypic Characterization of Miltefosine-Resistant Leishmania major

Trypanosomatid parasites of the genus Leishmania are the causative agents of leishmaniasis, a neglected tropical disease with several clinical manifestations. Leishmania major is the causative agent of cutaneous leishmaniasis (CL), which is largely characterized by ulcerative lesions appearing on the skin. Current treatments of leishmaniasis include pentavalent antimonials and amphotericin B, however, the toxic side effects of these drugs and difficulty with distribution makes these options less than ideal. Miltefosine (MIL) is the first oral treatment available for leishmaniasis. Originally developed for cancer chemotherapy, the mechanism of action of MIL in Leishmania spp. is largely unknown. While treatment with MIL has proven effective, higher tolerance to the drug has been observed, and resistance is easily developed in an in vitro environment. Utilizing stepwise selection we generated MIL-resistant cultures of L. major and characterized the fitness of MIL-resistant L. major. Resistant parasites proliferate at a comparable rate to the wild-type (WT) and exhibit similar apoptotic responses. As expected, MIL-resistant parasites demonstrate decreased susceptibility to MIL, which reduces after the drug is withdrawn from culture. Our data demonstrate metacyclogenesis is elevated in MIL-resistant L. major, albeit these parasites display attenuated in vitro and in vivo virulence and standard survival rates in the natural sandfly vector, indicating that development of experimental resistance to miltefosine does not lead to an increased competitive fitness in L. major.     

Corticoadrenal activity regulates in rat betaine-homocysteine <Emphasis Type="Italic">S</Emphasis>-methyltransferase expression with opposites effects in liver and kidney

Betaine-homocysteine S-methyltransferase (BHMT) is an enzyme that converts homocysteine (Hcy) to methionine using betaine as a methyl donor. Betaine also acts as osmolyte in kidney medulla, protecting cells from high extracellular osmolarity. Hepatic BHMT expression is regulated by salt intake. Hormones, particularly corticosteroids, also regulate BHMT expression in rat liver. We investigated to know whether the corticoadrenal activity plays a role in kidney BHMT expression. BHMT activity in rat kidneys is several orders of magnitude lower than in rat livers and only restricted to the renal cortex. This study confirms that corticosteroids stimulate BHMT activity in the liver and, for the first time in an animal model, also up-regulate the BHMT gene expression. Besides, unlike the liver, corticosteroids in rat kidney down-regulate BHMT expression and activity. Given that the classical effect of adrenocortical activity on the kidney is associated with sodium and water re-absorption by the distal tubule leading to volume expansion, by promoting lesser use of betaine as a methyl donor, corticosteroids would preserve betaine for its other role as osmoprotectant against changes in the extracellular osmotic conditions. We conclude that corticosteroids are, at least in part, responsible for the inhibition of BHMT expression and activity in rat kidneys.     

The development of cephalic armor in the tokay gecko (Squamata: Gekkonidae: Gekko gecko)

Armored skin resulting from the presence of bony dermal structures, osteoderms, is an exceptional phenotype in gekkotans (geckos and flap-footed lizards) only known to occur in three genera: Geckolepis, Gekko, and Tarentola. The Tokay gecko (Gekko gecko LINNAEUS 1758) is among the best-studied geckos due to its large size and wide range of occurrence, and although cranial dermal bone development has previously been investigated, details of osteoderm development along a size gradient remain less well-known. Likewise, a comparative survey of additional species within the broader Gekko clade to determine the uniqueness of this trait has not yet been completed. Here, we studied a large sample of gekkotans (38 spp.), including 18 specimens of G. gecko, using X-rays and high-resolution computed tomography for visualizing and quantifying the dermal armor in situ. Results from this survey confirm the presence of osteoderms in a second species within this genus, Gekko reevesii GRAY 1831, which exhibits discordance in timing and pattern of osteoderm development when compared with its sister taxon, G. gecko. We discuss the developmental sequence of osteoderms in these two species and explore in detail the formation and functionality of these enigmatic dermal ossifications. Finally, we conducted a comparative analysis of endolymphatic sacs in a wide array of gekkotans to explore previous ideas regarding the role of osteoderms as calcium reservoirs. We found that G. gecko and other gecko species with osteoderms have highly enlarged endolymphatic sacs relative to their body size, when compared to species without osteoderms, which implies that these membranous structures might fulfill a major role of calcium storage even in species with osteoderms.     

Expression and localization of endocrine gland-derived vascular endothelial growth factor (EG-VEGF) in human pancreas and pancreatic adenocarcinoma

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) was recently identified as the first tissue-specific angiogenic molecule. EG-VEGF (the gene product of PROK-1) appears to be expressed exclusively in steroid-producing organs such as the ovary, testis, adrenals and placenta. Since the human pancreatic cells retain steroidogenic activity, in the present study we ascertained whether this angiogenic factor is expressed in normal pancreas and pancreatic adenocarcinoma. Tissue samples from normal males (n=5), normal females (n=5) and from surgically resected adenocarcinomas (n=2) were processed for RT-PCR and immunohistochemical studies. Results from semi-quantitative analysis by RT-PCR suggest a distinct expression level for EG-VEGF in the different tissue samples. The relative amount of EG-VEGF mRNA in pancreas was more abundant in female adenocarcinoma (0.89) followed by male adenocarcinoma (0.71), than normal female (0.64) and normal male (0.38). The expression of mRNA for EG-VEGF in normal tissue was significantly higher in females than in males. All samples examined showed specific immunostaining for EG-VEGF. In male preparations, the positive labeling was localized predominantly within the pancreatic islets while in female preparations the main staining was detected towards the exocrine portion. Specific immunolabeling was also observed in endothelial cells of pancreatic blood vessels. Our data provide evidence that the human pancreas expresses the EG-VEGF, a highly specific mitogen which regulates proliferation and differentiation of the vascular endothelium. The significance of this finding could be interpreted as either, EG-VEGF is not exclusive of endocrine organs, or the pancreas should be considered as a functional steroidogenic tissue. The extent of the expression of EG-VEGF appears to have a dimorphic pattern in normal and tumoral pancreatic tissue.     

Modeling Susceptibility versus Resistance in Allergic Airway Disease Reveals Regulation by Tec Kinase Itk

Murine models of allergic asthma have been used to understand the mechanisms of development and pathology in this disease. In addition, knockout mice have contributed significantly to our understanding of the roles of specific molecules and cytokines in these models. However, results can vary significantly depending on the mouse strain used in the model, and in particularly in understanding the effect of specific knockouts. For example, it can be equivocal as to whether specific gene knockouts affect the susceptibility of the mice to developing the disease, or lead to resistance. Here we used a house dust mite model of allergic airway inflammation to examine the response of two strains of mice (C57BL/6 and BALB/c) which differ in their responses in allergic airway inflammation. We demonstrate an algorithm that can facilitate the understanding of the behavior of these models with regards to susceptibility (to allergic airway inflammation) (S_aai) or resistance (R_aai) in this model. We verify that both C57BL/6 and BALB/c develop disease, but BALB/c mice have higher S_aai for development. We then use this approach to show that the absence of the Tec family kinase Itk, which regulates the production of Th2 cytokines, leads to R_aai in the C57BL/6 background, but decreases S_aai on the BALB/c background. We suggest that the use of such approaches could clarify the behavior of various knockout mice in modeling allergic asthma.     

Exopolysaccharide, pigment and protein production by the marine microalga Chroomonas sp. in semicontinuous cultures

The marine microalga Chroomonas sp. isolated from Venezuela was grown in semicontinuous culture in order to study the effect of renewal rate and nutrient concentration on alloxanthin, chlorophyll a, carotenoid, carbohydrate, exopolysaccharide, protein and cell productivity. Maximal cell productivity of 8.43 ± 1.8 and 8.81 ± 2.3 × 10⁹ cell l^–1 day^–1 were achieved with renewal rates of 30 and 40%. Maximal protein and chlorophyll productivity of 64.64 ± 2.3 and 2.72 ± 0.3 mg l^–1 day^–1 were obtained with renewal rate of 20 and 30%. Biochemical composition of Chroomonas sp. was influenced by renewal rate. Nutrient concentration seems not to affect cell, protein, chlorophyll and carotenoid productivity. However, carbohydrate and exopolysaccharide productivity of 7.56 ± 0.4 and 9.57 ± 1.2 mg l^–1 day^–1 were increased at 12 mM NaNO₃(P < 0.05). Also, alloxanthin and chlorophyll a production analysed by HPLC, were higher between 8 and 12 mM NaNO₃ at a renewal rate of 30%. Results demonstrated that a renewal rate of 30% and nutrient concentration at 8 mM NaNO₃ optimize the cell, protein, carbohydrate, chlorophyll a, and exopolysaccharide productivity in semicontinuous cultures of Chroomonas. This microalga, as biological source of commercially valuable compounds, shows high capacity for changing its productivity and biochemical composition in semicontinuous system on the basis of nutrient concentration and the renewal rate.     

Temperature and pH/CO(2) modulate respiratory activity in the isolated brainstem of the bullfrog (Rana catesbeiana)

The effects of temperature and pH/CO(2) were examined in isolated brainstem preparations from adult North American bullfrogs (Rana catesbeiana). These experiments were undertaken to determine the effects of temperature on fictive breathing, central pH/CO(2) chemoreception, and to examine potential alphastat regulation of respiration in vitro. Adult bullfrog brainstem preparations were isolated, superfused with an artificial cerebrospinal fluid (aCSF) and respiratory-related neural activity was recorded from cranial nerves V, X and XII. In Series I experiments (N=8), brainstem preparations were superfused with aCSF equilibrated with 2% CO(2) at temperatures ranging from 10 to 30 degrees C. Neural activity was present in all preparations in the temperature range of 15-25 degrees C, but was absent in most preparations when aCSF was at 10 or 30 degrees C. The absence of fictive breathing at high (30 degrees C) temperatures was transient since fictive breathing could be restored upon returning the preparation to 20 degrees C. In Series II experiments (N=10), preparations were superfused with aCSF equilibrated with 0%, 2% and 5% CO(2) at temperatures of 15, 20 and 25 degrees C. Fictive breathing frequency (f(R)) was significantly dependent upon aCSF pH at all three temperatures, with slopes ranging from -0.82 min(-1) pH unit(-1) (15 degrees C) to -3.3 min(-1) pH unit(-1) (20 degrees C). There was a significant difference in these slopes (P<0.02), indicating that central chemoreceptor sensitivity increased over this temperature range. Fictive breathing frequency was significantly dependent upon the calculated alpha-imidazole (alpha(Im)) ionization (P<0.05), consistent with the alphastat hypothesis for the effects of temperature on the regulation of ventilation. However, most of the variation in f(R) was not explained by alpha(Im) (R(2)=0.05), suggesting that other factors account for the regulation of fictive breathing in this preparation. The results indicate that the in vitro brainstem preparation of adult bullfrogs has a limited temperature range (15-25 degrees C) over which fictive breathing is consistently active. Although there is a close correspondence of ventilation in vitro and in vivo at low temperatures, these data suggest that, as temperature increases, changes in ventilation in the intact animal are likely to be more dependent upon peripheral feedback which assumes a greater integrative role with respect to chemoreceptor drive, respiratory frequency and tidal volume.     

The conserved Xanthomonas campestris pv. vesicatoria effector protein XopX is a virulence factor and suppresses host defense in Nicotiana benthamiana

Nicotiana benthamiana leaves display a visible plant cell death response when infiltrated with a high titer inoculum of the non-host pathogen, Xanthomonas campestris pv. vesicatoria (Xcv). This visual phenotype was used to identify overlapping cosmid clones from a genomic cosmid library constructed from the Xcv strain, GM98-38. Individual cosmid clones from the Xcv library were conjugated into X. campestris pv. campestris (Xcc) and exconjugants were scored for an altered visual high titer inoculation response in N. benthamiana. The molecular characterization of the cosmid clones revealed that they contained a novel gene, xopX, that encodes a 74-kDa type III secretion system (TTSS) effector protein. Agrobacterium-mediated transient expression of XopX in N. benthamiana did not elicit the plant cell death response although detectable XopX protein was produced. Interestingly, the plant cell death response occurred when the xopX Agrobacterium-mediated transient expression construct was co-inoculated with strains of either XcvDeltaxopX or Xcc, both lacking xopX. The co-inoculation complementation of the plant cell death response also depends on whether the Xanthomonas strains contain an active TTSS. Transgenic 35S-xopX-expressing N. benthamiana plants also have the visible plant cell death response when inoculated with the non-xopX-expressing strains XcvDeltaxopX and Xcc. Unexpectedly, transgenic 35S-xopX N. benthamiana plants displayed enhanced susceptibility to bacterial growth of Xcc as well as other non-xopX-expressing Xanthomonas and Pseudomonas strains. This result is also consistent with the increase in bacterial growth on wild type N. benthamiana plants observed for Xcc when XopX is expressed in trans. Furthermore, XopX contributes to the virulence of Xcv on host pepper (Capsicum annuum) and tomato (Lycopersicum esculentum) plants. We propose that the XopX bacterial effector protein targets basic innate immunity in plants, resulting in enhanced plant disease susceptibility.     

Assembly of truncated HCV core antigen into virus-like particles in Escherichia coli

Core protein is one of the most conserved and immunogenic of the hepatitis C virus proteins. Several pieces of experimental evidence suggest its ability for formation of virus like particles alone or in association with other viral proteins in mammalian or yeast cells with great similarity to those detected in patient sera and liver extract. In this work we report an Escherichia coli-derived truncated hepatitis C core protein that is able to aggregate. SDS-PAGE and size exclusion chromatography patterns bring to mind the aggregation of monomers of recombinant protein Co.120. The Co.120 protein migrated with buoyant density of 1.28 g/cm(3) when analyzed using CsCl density gradient centrifugation. Spherical structures with an average diameter of 30 nm were observed using electron microscopy. We report here that VLPs are generated when the first 120 aa of HCV core protein are expressed in E. coli.     

Intracellular distribution of lysosomal sialidase is controlled by the internalization signal in its cytoplasmic tail

Sialidase (neuraminidase), encoded by the neu-1 gene in the major histocompatibility complex locus catalyzes the intralysosomal degradation of sialylated glycoconjugates. Inherited deficiency of sialidase results in sialidosis or galactosialidosis, both severe metabolic disorders associated with lysosomal storage of oligosaccharides and glycopeptides. Sialidase also plays an important role in cellular signaling and is specifically required for the production of cytokine interleukin-4 by activated T lymphocytes. In these cells, neu-1-encoded sialidase activity is increased on the cell surface, suggesting that a specific mechanism regulates sorting of this enzyme to the plasma membrane. We investigated that mechanism by first showing that sialidase contains the internalization signal found in lysosomal membrane proteins targeted to endosomes via clathrin-coated pits. The signal consists of a C-terminal tetrapeptide (412)YGTL(415), with Tyr(412) and Leu(415) essential for endocytosis of the enzyme. We further demonstrated that redistribution of sialidase from lysosomes to the cell surface of activated lymphocytes is accompanied by increased reactivity of the enzyme with anti-phosphotyrosine antibodies. We speculate that phosphorylation of Tyr(412) results in inhibition of sialidase internalization in activated lymphocytes.