Babesia bigemina sexual stages are induced in vitro and are specifically recognized by antibodies in the midgut of infected Boophilus microplus ticks

Babesia bigemina, a causative agent of bovine babesiosis, is transmitted from one bovine to another only by infected ticks. The life cycle of B. bigemina includes a sexual phase in the tick host; however, molecules from sexual stages of any Babesia species have not been characterized. This is the first report of the induction of sexual stages of any Babesia species in vitro, free of tick antigens. Intraerythrocytic parasites were cultured in vitro for 20h using an induction medium. Extraerythrocytic parasites were first seen 3h post induction; elongated stages with long projections appeared at 6h post induction and by 9h they paired and fused to form larger stages. Round zygotes appeared 20h post induction. Moreover, by using Percoll gradients, sexual stages were purified free of contaminating intraerythrocytic stages. Purified parasites were used to generate polyclonal antibodies, which specifically bound to antigens expressed in sexual stages induced in vitro, but not to antigens expressed in intraerythrocytic stages. Importantly, these antibodies specifically identified sexual stages from midguts of female Boophilus microplus ticks fed on infected cattle.     

Aven and Bcl-xL enhance protection against apoptosis for mammalian cells exposed to various culture conditions

A balance between proliferation and cell death is critical for achieving desirable high cell densities in mammalian cell culture. In this study, we evaluate a recently discovered anti-apoptotic gene, aven, and examine its effectiveness alone and in combination with a member of the Bcl-2 family, bcl-xL. The commercially popular cell line, Chinese hamster ovary (CHO), was genetically modified to constitutively express aven, bcl-xL, and the two genes in combination. Cells were exposed to several model insults that simulate severe bioreactor environments, including serum deprivation, spent medium, and Sindbis virus infection, as well as staurosporine, a known chemical inducer of apoptosis. CHO cells exhibited DNA fragmentation, a hallmark of apoptosis, after exposure to these model insults. After exposure to serum deprivation, 4- and 5-day spent medium, and staurosporine, cells expressing Aven provided limited protection against cell death when compared with the protection afforded by cells expressing Bcl-xL alone. However, the highest survival levels for all insults were achieved when Aven was expressed in combination with Bcl-xL. In fact, Aven appeared to act synergistically to enhance the protective function of Bcl-xL for several insults, because the protective function of the two genes expressed together in one cell line often exceeded the additive protective levels of each anti-apoptosis gene expressed alone. Surprisingly, Aven expression provided a mildly pro-apoptotic response in CHO isolates infected with Sindbis virus. However, CHO cells expressing both Bcl-xL and Aven showed protection against Sindbis virus infection due to the inhibitory properties of the bcl-xL anti-apoptosis gene. This study shows that combinatorial anti-apoptosis cell engineering strategies may be the most effective mechanisms for providing extended protection against cell death in mammalian cell culture.     

Genetic diversity and insecticide resistance of Myzus persicae (Hemiptera: Aphididae) populations from tobacco in Chile: evidence for the existence of a single predominant clone

The tobacco-feeding race of Myzus persicae (Sulzer), formerly known as M. nicotianae Blackman, was introduced into Chile during the last decade. In order to evaluate the genetic diversity and insecticide resistance status of Chilean tobacco aphid populations, a field survey was conducted in 35 tobacco fields covering a 300 km latitudinal survey. The populations sampled were characterized using microsatellite markers and morphometric multivariate analysis. Insecticide resistance levels were assessed through a microplate esterase assay and the mutation status of the kdr gene. All samples collected corresponded to the same anholocyclic aphid genotype, and showed morphological variation within the range expected for the tobacco-feeding race of M. persicae. Esterase activity showed the level and variability expected for an R1 clone lacking mutations in the sodium channels (susceptible kdr), thus corresponding to a type slightly resistant to organophosphate and carbamate, and susceptible to pyrethroid insecticides.     

Ontogeny and effect of cortisol on vasopressin-1b receptor expression in anterior pituitaries of fetal sheep

Corticotroph responsiveness to arginine vasopressin (AVP) increases during late gestation in fetal sheep. The mechanism of this increase in AVP responsiveness is currently unknown but could be related to an increase in vasopressin type 1b (V1b) receptor expression in the pituitary during development. To determine if there are ontogenic changes in V1b receptor expression that may help explain the changes in ACTH responses to AVP, we studied pituitaries from three groups of fetal sheep [100, 120, or 140 days gestational age (dGA)]. V1b receptor mRNA and protein significantly decreased by 140 dGA. Peak V1b mRNA levels were detected at 100 dGA, while peak V1b protein levels were detected at 120 dGA. The reduction in V1b receptor expression in late gestation may be due to the naturally occurring peripartum increase in fetal plasma cortisol because cortisol infusion at 122-130 dGA decreased V1b receptor mRNA. Thus there is a marked decrease in the expression of the V1b receptor in the pituitary during fetal development, leaving the role of the V1b receptor in increasing AVP responsiveness uncertain.     

Identification of chemokines and a chemokine receptor in cichlid fish,shark, and lamprey

Chemokines are small, inducible, structurally related proteins that guide cells expressing the right chemokine receptors to sites of immune response. They have been identified and studied extensively in mammals, but little is known about their presence in other vertebrate groups. Here we describe seven new chemokines in bony fish and one in a cartilaginous fish, as well as one chemokine receptor in a jawless vertebrate. All eight chemokines belong to the SCYA (CC) subfamily characterized by four conserved cysteine residues of which the first two are adjacent. The chemokine receptor is of the CXCR4 type. Phylogenetic analysis does not reveal any clear evidence of orthology of fish and human chemokines. Although the divergence of the subfamilies began before the fish-tetrapod split, much of the divergence within the subfamilies took place separately in the two vertebrate groups. The existence of a chemokine receptor in the lamprey indicates that chemokines are apparently also present in the Agnatha.     

Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS

Recent studies indicated that there is a distinct mitochondrial nitric oxide synthase (mtNOS) enzyme, which may be identical to the other known NOS isoforms. We investigated the possible involvement of the endothelial, the neuronal, and the inducible NOS isoforms (eNOS, nNOS, iNOS, respectively) in mitochondrial NO production. Mouse liver mitochondria were prepared by Percoll gradient purification from wild-type and NOS knockout animals. NOS activity was measured by the arginine conversion assay, NO production of live mitochondria was visualized by the fluorescent probe DAF-FM with confocal microscopy and measured with flow cytometry. Western blotting or immunoprecipitation was performed with 12 different anti-NOS antibodies. Mitochondrial NOS was purified by arginine, 2,5 ADP and calmodulin affinity columns. We observed NO production and NOS activity in mitochondria, which was not attenuated by classic NOS inhibitors. We also detected low amounts of eNOS protein in the mitochondria, however, NO production and NOS activity were intact in eNOS knockout animals. Neither nNOS nor iNOS were present in the mitochondria. Furthermore, we could not find mitochondrial targeting signals in the sequences of either NOS proteins. Taken together, the presented data do not support the hypothesis that any of the known NOS enzymes are present in the mitochondria in physiologically relevant levels.     

The phylogenetic relationship of tetrapod, coelacanth, and lungfish revealed by the sequences of forty-four nuclear genes

The origin of tetrapods is a major outstanding issue in vertebrate phylogeny. Each of the three possible principal hypotheses (coelacanth, lungfish, or neither being the sister group of tetrapods) has found support in different sets of data. In an attempt to resolve the controversy, sequences of 44 nuclear genes encoding amino acid residues at 10,404 positions were obtained and analyzed. However, this large set of sequences did not support conclusively one of the three hypotheses. Apparently, the coelacanth, lungfish, and tetrapod lineages diverged within such a short time interval that at this level of analysis, their relationships appear to be an irresolvable trichotomy.     

p34cdc2-mediated phosphorylation mobilizes microtubule-organizing centers from the apical intermediate filament scaffold in CACO-2 epithelial cells

We have shown previously that centrosomes and other microtubule-organizing centers (MTOCs) attach to the apical intermediate filament (IF) network in CACO-2 cells. In this cell line, intermediate filaments do not disorganize during mitosis. Therefore, we speculated that the trigger of the G(2)-M boundary may also detach MTOCs from their IF anchor. If that was the case, at least one of the proteins involved in the attachment must be phosphorylated by p34(cdc2) (cdk1). Using confocal microscopy and standard biochemical analysis, we found that p34(cdc2)-mediated phosphorylation indeed released MTOCs from IFs in permeabilized cells. In isolated, immunoprecipitated multiprotein complexes containing both gamma-tubulin and cytokeratin 19, p34(cdc2) phosphorylated only one protein, and phosphorylation released cytokeratin 19 from the complexes. We conclude that this as yet unidentified protein is a part of the molecular mechanism that attaches MTOCs to IFs in interphase.     

Light quality effect on the seeds germination in tropical pioneer tree Heliocarpus appendiculatus (Tiliaceae)

The objective of this study was to determine whether seeds of the pioneer tree Heliocarpus appendiculatus possess photoblastic dormancy. Seeds from nine trees were collected in Los Tuxtlas, Mexico. In order to test for the presence of photoblastic dormancy, germination experiments were carried out separately on seeds of each individual tree. The seeds from each tree were sown and subjected to four light treatments: fluorescent white light, red light (660 nm), far red light (730 nm), and darkness. A total of 50 seeds were sown in each plastic Petri dish (three replicates per treatment) on an agar solution. Experiments were carried out at a constant temperature of 20 degrees C, and a 12:12 hr (L:D) photoperiod. In addition, seeds of three individuals were sown on agar and subjected to a light quality gradient from red to far red (1.1-0.2). Results show that final germination percentages of seeds were unaffected by light quality in all individuals. Nevertheless, germination was delayed by 24 hr in the seeds of four individuals under the far red light treatment. By the end of fourth day, final germination did not differ among treatments. Further, germination of the three individuals under the red/far red gradient was unaffected. Seeds of H. appendiculatus lack photoblastic dormancy and germination behavior can not be used to explain the absence of seedlings below the canopy. We propose that this absence is due to the failure of the seedlings to establish themselves under the canopy.