Hormonal regulation of apoptosis in the endometrium of common marmosets (Callithrix jacchus)

Phase-dependent apoptotic changes in the human endometrium during an ovarian cycle imply a potential role of steroids in the regulation of apoptosis. The present study was undertaken to determine the direct role of hormones in endometrial apoptosis in marmosets (Callithrix jacchus), a primate species which shows similarity to humans in terms of the cycle length and pattern. Endometrial apoptosis was detected by 3'-end labeling (TUNEL) in various phases of ovarian cycle in naturally cycling healthy marmosets (n=14) and also in ovariectomized marmosets (n=13) treated with either estradiol alone (E) or progesterone alone (P) or estradiol followed by progesterone (E+P). Expressions of apoptosis associated genes such as Bcl-2 family members (Bax and Bcl-2), proliferating cell nuclear antigen (PCNA)--a proliferation marker and steroid receptors, ERalpha and PR A were analysed by immunohistochemical methods. Apoptosis was intense in the glandular epithelial cells of endometrium during the mid-luteal phase as compared to other phases in naturally cycling animals; in the E+P group as compared to other groups of ovariectomized animals (P<0.05). Pronounced apoptosis in the mid-luteal phase was accompanied by the increased expression of Bax in glandular epithelial cells; while Bcl-2 immunoreactivity remained unchanged. PCNA expression was higher in the naturally cycling animals in the follicular phase and in the E group of the ovariectomized animals as compared those in the other groups. Immunoreactive ERalpha and PR A in glandular epithelial cells were most abundant during early follicular phase in naturally cycling animals and in both E and E+P groups among the ovariectomized animals. The present study highlights the importance of apoptosis in endometrial remodeling during the ovarian cycle and secondly, the role of both estradiol and progesterone in the regulation of apoptosis.     

Application of XANES spectroscopy in understanding the metabolism of selenium in isolated rainbow trout hepatocytes: insights into selenium toxicity

Selenium (Se) is an essential element, but causes toxic effects in fish at a slightly elevated level beyond the threshold. However, the degree of Se toxicity differs depending on the chemical forms of Se (e.g., organic vs. inorganic) to which fish are exposed to. The mechanisms of Se metabolism and toxicity in fish, particularly at cellular level, are poorly understood. The present study was designed to examine the metabolic fate of different seleno-compounds, both inorganic and organic, in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss) in primary culture using XANES spectroscopy. In cells exposed to 100 μM of selenate and selenite for 6-24 h, elemental Se was found to be the primary metabolite. Whereas, selenocystine appeared to be the major metabolite in cells exposed to 100 μM seleno-L-methionine for 6-24 h. Interestingly, we recorded L-methionine-γ-lyase activity in S9 fraction of cell lysate-an enzyme that directly catalyzes selenomethionine into methylselenol. We also found concurrent reduction of glutathione (GSH) concentration following reaction of seleno-L-methionine with cellular S9 fraction. Moreover, we observed a rapid increase in cellular reactive oxygen species (ROS) generation with increasing seleno-L-methionine exposure dose (100-1000 μM). These findings indicated the rapid cellular metabolism of seleno-L-methionine into methylselenol at higher exposure dose (≥100 μM), and the occurrence of GSH mediated redox cycling of methylselenol--a process that is known to produce reactive oxygen species (ROS). Overall, our results suggest that inorganic and organic selenium are metabolized through different metabolic pathways in rainbow trout hepatocytes. The findings of our study have important implications for understanding the chemical species-specific differences in Se toxicity to fish.     

Lysozyme in egg whites of tortoises and turtle. Purification and properties of egg white lysozyme of Trionyx gangeticus Cuvier.

Egg white proteins of three species of tortoises and turtle and of hen have been compared by electrophoretic and immunochemical methods. The proteins lacked similarity in electrophoresis, but tortoise and turtle egg white proteins which did not crossreact with those of the hen showed some cross-reaction among themselves. The occurrence of lysozyme as two allelic variants which were distinguishable in electrophoresis was noted only in the egg white of one of the species of tortoise, namely, Trionyx gangeticus Cuvier. Tortoise lysozyme which showed strong lytic activity toward cell walls of Micrococcus lysodeikticus did not exhibit any cross-reaction with hen lysoyzme. It was purified, crystallized, and found to be homogeneous in sodium dodecyl sulfatepolyacrylamide gel electrophoresis, immunochemical tests, and sedimentation. The physicochemical and enzymatic properties of tortoise lysozyme were found to be strikingly similar to those of hen lysozyme with minor differences which could be due to differences in their primary structure. Its average molecular weight of 15,400 was determined from sedimentation and diffusion coefficient values, Archibald experiment, and amino acid composition. The molecule appeared to undergo pH-dependent expansion at pH 2 and dimerization above pH 5.7. In enzymatic properties, tortoise lysozyme showed a specific activity of 29,000–31,000 units and gave a pH optimum at pH 7.5 and an apparent K_a value of 250 mg· liter^?1. Like hen lysozyme, its activity showed strong ionic strength dependence, weak chitinase activity, susceptibility to inhibition by N-acetyl-glucosamine, and stability toward heat.     

Grape proanthocyanidins induce apoptosis by loss of mitochondrial membrane potential of human non-small cell lung cancer cells in vitro and in vivo

Lung cancer remains the leading cause of cancer-related deaths worldwide, and non-small cell lung cancer (NSCLC) represents approximately 80% of total lung cancer cases. The use of non-toxic dietary phytochemicals can be considered as a chemotherapeutic strategy for the management of the NSCLC. Here, we report that grape seed proanthocyanidins (GSPs) induce apoptosis of NSCLC cells, A549 and H1299, in vitro which is mediated through increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspases 9, 3 and poly (ADP-ribose) polymerase (PARP). Pre-treatment of A549 and H1299 cells with the caspase-3 inhibitor (z-DEVD-fmk) significantly blocked the GSPs-induced apoptosis of these cells confirmed that GSPs-induced apoptosis is mediated through activation of caspases-3. Treatments of A549 and H1299 cells with GSPs resulted in an increase in G1 arrest. G0/G1 phase of the cell cycle is known to be controlled by cyclin dependent kinases (Cdk), cyclin-dependent kinase inhibitors (Cdki) and cyclins. Our western blot analyses showed that GSPs-induced G1 cell cycle arrest was mediated through the increased expression of Cdki proteins (Cip1/p21 and Kip1/p27), and a simultaneous decrease in the levels of Cdk2, Cdk4, Cdk6 and cyclins. Further, administration of 50, 100 or 200 mg GSPs/kg body weight of mice by oral gavage (5 d/week) markedly inhibited the growth of s.c. A549 and H1299 lung tumor xenografts in athymic nude mice, which was associated with the induction of apoptotic cell death, increased expression of Bax, reduced expression of anti-apoptotic proteins and activation of caspase-3 in tumor xenograft cells. Based on the data obtained in animal study, human equivalent dose of GSPs was calculated, which seems affordable and attainable. Together, these results suggest that GSPs may represent a potential therapeutic agent for the non-small cell lung cancer.     

The Effect of Assortative Mating on the Coexistence of a Hybridogenetic Waterfrog and Its Sexual Host

In central Europe, the hybridogenetic waterfrog Rana esculenta, a hybrid between Rana ridibunda and Rana lessonae, lives in sympatry with one of its parental species, the poolfrog Rana lessonae. As R. esculenta has to backcross constantly with R. lessonae in order to produce viable offspring, this coexistence is obligatory for R. esculenta. Since R. esculenta has a higher primary fitness than R. lessonae, a mechanism is required that prevents the hybrid from driving the parental species, and hence itself, to extinction. Here, we present an analytical model and a computer simulation that investigate whether assortative mating can operate as a such a control mechanism. Our results show that assortative mating is very effective in regulating coexistence in such a hybrid-host system. This is particularly true when choice is affected by the proportion of the two male types in the population. Furthermore, we could show that even if the species composition in a mixed hybrid-host population may be largely influenced by differences in life-history parameters, assortative mating still plays a very important role by stabilizing coexistence. Thus, mating behavior turns out to be more important for the populations dynamics of hybridogenetic waterfrog systems than previously assumed.     

Controlled delivery of paclitaxel from stent coatings using poly(hydroxystyrene-b-isobutylene-b-hydroxystyrene) and its acetylated derivative

A poly(styrene-b-isobutylene-b-styrene) (SIBS) triblock polymer is employed as the polymer drug carrier for the TAXUS Express2 Paclitaxel-Eluting Coronary Stent system (Boston Scientific Corp.). It has been shown that the release of paclitaxel (PTx) from SIBS can be modulated by modification of either drug-loading ratio or altering the triblock morphology by blending. In the present work, results toward achieving release modulation of PTx by chemical modification of the styrenic portion (using hydroxystyrene or its acetylated version) of the SIBS polymer system are reported. The synthesis of the precursor poly[(p-tert-butyldimethylsilyloxystyrene)]-b-isobutylene-b-[(p-tert-butyldimethylsilyloxystyrene] triblock copolymers was accomplished by living sequential block copolymerization of isobutylene (IB) and p-(tert-butyldimethylsiloxy)styrene (TBDMS) utilizing the capping-tuning technique in a one-pot procedure in methylcyclohexane/CH3Cl at -80 degrees C. This procedure involved the living cationic polymerization of IB with the 5-tert-butyl-1,3-bis(1-chloro-1-methylethyl)benzene/TiCl4 initiating system and capping of living difunctional polyisobutylene (PIB) chain ends with 1,1-ditolylethylene (DTE) followed by addition of titanium(IV) isopropoxide (Ti(OIp)4) to lower the Lewis acidity before the introduction of TBDMS. Deprotection of the product with tetrabutylammonium fluoride yielded poly(hydroxystyrene-b-isobutylene-b-hydroxystyrene), which was quantitatively acetylated to obtain the acetylated derivative. The hydroxystyrene and acetoxystyrene triblock copolymers have acceptable mechanical properties for use as drug delivery coatings for coronary stent applications. It was concluded that the hydrophilic nature of the endblocks and polarity effects on the drug/polymer miscibility lead to enhanced release of PTx from these polymers. The drug-polymer miscibility was confirmed by differential scanning calorimetry and atomic force microscopy evaluations.     

Antitubercular nitrofuran isoxazolines with improved pharmacokinetic properties

A series of tetracyclic nitrofuran isoxazoline anti-tuberculosis agents was designed and synthesized to improve the pharmacokinetic properties of an initial lead compound, which had potent anti-tuberculosis activity but suffered from poor solubility, high protein binding and rapid metabolism. In this study, structural modifications were carried on the outer phenyl and piperidine rings to introduce solubilizing and metabolically blocking functional groups. The compounds generated were evaluated for their in vitro antitubercular activity, bacterial spectrum of activity, solubility, permeability, microsomal stability and protein binding. Pharmacokinetic profiles for the most promising candidates were then determined. Compounds with phenyl morpholine and pyridyl morpholine outer rings were found to be the most potent anti-tuberculosis agents in the series. These compounds retained a narrow antibacterial spectrum of activity, with weak anti-Gram positive and no Gram negative activity, as well as good activity against non-replicating Mycobacterium tuberculosis in a low oxygen model. Overall, the addition of solubilizing and metabolically blocked outer rings did improve solubility and decrease protein binding as designed. However, the metabolic stability for compounds in this series was generally lower than desired. The best three compounds selected for in vivo pharmacokinetic testing all showed high oral bioavailability, with one notable compound showing a significantly longer half-life and good tolerability supporting its further advancement.     

Proteome Analysis of Plasmodium falciparum Extracellular Secretory Antigens at Asexual Blood Stages Reveals a Cohort of Proteins with Possible Roles in Immune Modulation and Signaling

The highly co-evolved relationship of parasites and their hosts appears to include modulation of host immune signals, although the molecular mechanisms involved in the host-parasite interplay remain poorly understood. Characterization of these key genes and their cognate proteins related to the host-parasite interplay should lead to a better understanding of this intriguing biological phenomenon. The malaria agent Plasmodium falciparum is predicted to export a cohort of several hundred proteins to remodel the host erythrocyte. However, proteins actively exported by the asexual intracellular parasite beyond the host red blood cell membrane (before merozoite egress) have been poorly investigated so far. Here we used two complementary methodologies, two-dimensional gel electrophoresis/MS and LC-MS/MS, to examine the extracellular secreted antigens at asexual blood stages of P. falciparum. We identified 27 novel antigens exported by P. falciparum in the culture medium of which some showed clustering with highly polymorphic genes on chromosomes, suggesting that they may encode putative antigenic determinants of the parasite. Immunolocalization of four novel secreted proteins confirmed their export beyond the infected red blood cell membrane. Of these, preliminary functional characterization of two novel (Sel1 repeat-containing) parasite proteins, PfSEL1 and PfSEL2 revealed that they down-regulate expression of cell surface Notch signaling molecules in host cells. Also a novel protein kinase (PfEK) and a novel protein phosphatase (PfEP) were found to, respectively, phosphorylate/dephosphorylate parasite-specific proteins in the extracellular culture supernatant. Our study thus sheds new light on malaria parasite extracellular secreted antigens of which some may be essential for parasite development and could constitute promising new drug targets.Plasmodium falciparum is a wide spread protozoan parasite responsible for over a million deaths annually mainly among children in sub-Saharan Africa (1). Like other apicomplexan parasites such as Leishmania, Trypanosoma, and Toxoplasma, Plasmodia depend on a series of intricate and highly evolved adaptations that enable them to evade destruction by the host immune responses. These protozoan parasites have provided some of the best leads in elucidating the mechanisms to circumvent innate immunity and adaptive humoral and cellular immunity (2). Ingenious strategies to escape innate defenses include subversion of attack by humoral effector mechanisms such as complement lysis and lysis by other serum components (3), remodeling of phagosomal compartments in which they reside (4), modulation of host cell signaling pathways (5), and modification of the antigen-presenting and immunoregulatory functions of dendritic cells, which provide a crucial link with the adaptive immune response (6). Malaria parasites also predominantly use antigenic diversity and clonal antigenic variation to evade adaptive immunity of the host (7). Surface-associated and secreted parasite proteins are major players in host-parasite cross-talk and are advantageously used by the parasite to counter the host immune system. Proteins secreted by a wide range of parasitic pathogens into the host microenvironment result in symptomatic infections. For example, the excretory-secretory (ES)¹ products of the parasitic fluke Fasciola hepatica are key players in host-parasite interactions (8). Among the apicomplexans, proteomics analyses of rhoptry organelles of Toxoplasma gondii have revealed many novel constituents of host-parasite interactions (9).The identification and trafficking of Plasmodium proteins exported into the host erythrocyte have been subjects of recent detailed investigations. A number of studies have identified Plasmodium proteins that contain signature sequence motifs, the host cell targeting signal or the Plasmodium export element (PEXEL), that target these proteins into the infected erythrocytes (10, 11). Recent proteomics analyses have identified novel proteins in the raftlike membranes of the parasite and on the surface of infected erythrocytes (12, 13). P. falciparum translationally controlled tumor protein (PfTCTP), a homolog of the mammalian histamine-releasing factor, has been shown to be released into the culture supernatant from intact as well as ruptured infected RBCs and causes histamine release from human basophils and IL-8 secretion from eosinophils (14). However, the total spectrum of proteins actively exported by the asexual intracellular parasite beyond the host RBC membrane (before merozoite egress) has been poorly investigated so far.In the present study, we used two complementary methodologies, two-dimensional gel electrophoresis (2DE)/MS and LC-MS/MS to examine the cohort of extracellular secreted antigens (ESAs) at asexual blood stages of P. falciparum. Our findings reveal that malaria parasites secrete a number of effector molecules such as immunomodulators and signaling proteins that are potentially involved in host-parasite interactions. Prominent among these are proteins with Sel1 domain, a protein of the LCCL family, a novel protein kinase, and a novel protein phosphatase. Secreted-extracellular/iRBC surface localization of some of these proteins was validated by immunolocalization studies. We also characterized the functions of some of these proteins in the culture supernatant, thus providing an insight into the nature of some of the malaria parasite extracellular antigens.     

Inter- and intra-strain variation in the 5.8S ribosomal RNA and internal transcribed spacer sequences of Entamoeba histolytica and comparison with Entamoeba dispar, Entamoeba moshkovskii and Entamoeba invadens

The ribosomal RNA genes in Entamoeba histolytica are located on circular DNA molecules in about 200 copies per genome equivalent. Nucleotide sequence analysis of the 5.8S rRNA gene and the flanking internal transcribed spacers was carried out to determine the degree of sequence divergence in the multiple rRNA gene copies of a given strain; amongst three different E. histolytica strains (HM-1:IMSS, Rahman and HK-9); and amongst four species of Entamoeba (Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii and Entamoeba invadens). The results show that all rRNA gene copies of a given strain are identical. Few nucleotide positions varied between strains of a species but the differences were very pronounced amongst species. In general, the internal transcribed spacer 2 sequence was more variable and may be useful for strain- and species-identification. The 5.8S rRNA gene and the internal transcribed spacer 2 of E. invadens were unusually small in size.