PAURODENDRON,A RHIZOMORPHIC LYCOPOD

From middle Pennsylvanian coal balls collected in Kansas and Illinois the ligules and rhizomorph of Paurodendron have been illustrated. Assignment of Paurodendron to the Lycopsida is corroborated. Discovery of the root-bearing portion of this Carboniferous lycopod establishes a diminutive, phyletic line in the Paleozoic with rhizomorphs smaller than many species of Isoeles. Paurodendron resembles the selaginelloid line more closely than other rhizomorphic types. Anatomical comparisons of Paurodendron with roots and root-bearing structures of lycopods are made; the root-bearing and a representative leaf-bearing portion of Paurodendron fraiponti have been reconstructed. The specific diagnosis has been emended, and the three species described by Fry have been reduced to one.     

Influence of Heparin Mimetics on Assembly of the FGF·FGFR4 Signaling Complex

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM₆), octasaccharide (HM₈), and decasaccharide (HM₁₀), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM₈ and HM₁₀ are significantly more potent than HM₆ in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM₈ relative to HM₆ in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM₈ to bind and dimerize FGF2. Notably FGF2·HM₈ exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.     

Drought constraints on C4 photosynthesis: stomatal and metabolic limitations in C3 and C4 subspecies of Alloteropsis semialata

The C4 photosynthetic pathway uses water more efficiently than the C3 type, yet biogeographical analyses show a decline in C4 species relative to C3 species with decreasing rainfall. To investigate this paradox, the hypothesis that the C4 advantage over C3 photosynthesis is diminished by drought was tested, and the underlying stomatal and metabolic mechanisms of this response determined. The effects of drought and high evaporative demand on leaf gas exchange and photosynthetic electron sinks in C3 and C4 subspecies of the grass Alloteropsis semialata were examined. Plant responses to climatic variation and soil drought were investigated using a common garden experiment with well-watered and natural rainfall treatments, and underlying mechanisms analysed using controlled drying pot experiments. Photosynthetic rates were significantly higher in the C4 than the C3 subspecies in the garden experiment under well-watered conditions, but this advantage was completely lost during a rainless period when unwatered plants experienced severe drought. Controlled drying experiments showed that this loss was caused by a greater increase in metabolic, rather than stomatal, limitations in C4 than in the C3 leaves. Decreases in CO2 assimilation resulted in lower electron transport rates and decreased photochemical efficiency under drought conditions, rather than increased electron transport to alternative sinks. These findings suggest that the high metabolic sensitivity of photosynthesis to severe drought seen previously in several C4 grass species may be an inherent characteristic of the C4 pathway. The mechanism may explain the paradox of why C4 species decline in arid environments despite high water-use efficiency.     

Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of infected host cells

Intracellular pathogens have evolved a wide array of mechanisms to invade and co-opt their host cells for intracellular survival. Apicomplexan parasites such as Toxoplasma gondii employ the action of unique secretory organelles named rhoptries for internalization of the parasite and formation of a specialized niche within the host cell. We demonstrate that Toxoplasma gondii also uses secretion from the rhoptries during invasion to deliver a parasite-derived protein phosphatase 2C (PP2C-hn) into the host cell and direct it to the host nucleus. Delivery to the host nucleus does not require completion of invasion, as evidenced by the fact that parasites blocked in the initial stages of invasion with cytochalasin D are able to target PP2C-hn to the host nucleus. We have disrupted the gene encoding PP2C-hn and shown that PP2C-hn-knockout parasites exhibit a mild growth defect that can be rescued by complementation with the wild-type gene. The delivery of parasite effector proteins via the rhoptries provides a novel mechanism for Toxoplasma to directly access the command center of its host cell during infection by the parasite.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain

Riboswitches are cis-acting genetic regulatory elements found commonly in bacterial mRNAs that consist of a metabolite-responsive aptamer domain coupled to a regulatory switch. Purine riboswitches respond to intracellular concentrations of either adenine or guanine/hypoxanthine to control gene expression. The aptamer domain of the purine riboswitch contains a pyrimidine residue (Y74) that forms a Watson-Crick base-pairing interaction with the bound purine nucleobase ligand that discriminates between adenine and guanine. We sought to understand the structural basis of this specificity and the mechanism of ligand recognition by the purine riboswitch. Here, we present the 2,6-diaminopurine-bound structure of a C74U mutant of the xpt-pbuX guanine riboswitch, along with a detailed thermodynamic and kinetic analysis of nucleobase recognition by both the native and mutant riboswitches. These studies demonstrate clearly that the pyrimidine at position 74 is the sole determinant of purine riboswitch specificity. In addition, the mutant riboswitch binds adenine and adenine derivatives well compared with the guanine-responsive riboswitch. Under our experimental conditions, 2,6-diaminopurine binds the RNA with DeltaH=-40.3 kcal mol(-1), DeltaS=-97.6 cal mol(-1)K(-1), and DeltaG=-10.73 kcal mol(-1). A kinetic determination of the slow rate (0.15 x 10(5)M(-1)s(-1) and 2.1 x 10(5)mM(-1)s(-1) for 2-aminopurine binding the adenine-responsive mutant riboswitch and 7-deazaguanine-binding guanine riboswitch, respectively) of association under varying experimental conditions allowed us to propose a mechanism for ligand recognition by the purine riboswitch. A conformationally dynamic unliganded state for the binding pocket is stabilized first by the Watson-Crick base pairing between the ligand and Y74, and by the subsequent ordering of the J2/3 loop, enclosing the ligand within the three-way junction.     

A molecular dissection of the glycoprotein hormone receptors

In glycoprotein hormone receptors, a subfamily of rhodopsin-like G protein-coupled receptors, the recognition and activation steps are carried out by separate domains of the proteins. Specificity of recognition of the hormones thyrotropin (TSH), lutropin (LH), human chorionic gonadotropin (hCG) and follitropin (FSH) involves leucine-rich repeats (LRRs) present in an N-terminal ectodomain, and can be associated with a limited number of residues at key positions of the LRRs. The mechanism by which binding of the hormones results in activation is proposed to involve switching of the ectodomain from a tethered inverse agonist to a full agonist of the serpentine, rhodopsin-like region of the receptor. Unexpectedly, the picture is complicated by the observation that promiscuous activation of one of the receptors (FSHr) by hCG or TSH can result from activating mutations affecting the serpentine region of the receptors.     

DNA and protein content of mouse sperm: Implications regarding sperm chromatin structure

A variety of biochemical and histochemical techniques have been used to compare the composition of chromatin in sperm nuclei isolated from the epididymides of five mouse strains. The DNA content was determined by phosphorus analysis, deoxyribose analysis, absorption spectroscopy at 260 nm, and cytomorphometry following gallocyanine chrome alum staining. All four methods indicate that the mouse sperm nucleus contains approx. 3.3 pg DNA and that the DNA content does not vary significantly among the strains tested. Three different techniques, quantitative amino acid analysis, absorption spectroscopy at 230 nm, and sperm head density analysis in cesium chloride, were used to determine the protein content. Sperm nuclei from each strain of mouse were found to have a protein to DNA ratio of 0.9 and a chromatin protein content of 3 pg/nucleus. Comparisons of the basic proteins by disc gel electrophoresis demonstrate that the sperm nuclei contain only protamine and lack significant levels of somatic histones or transition proteins. The sperm from each strain contained both mouse protamine variants and the relative distribution of the two proteins did not appear to differ among strains. Using this information, we have been able to draw certain conclusions regarding DNA-protamine interactions and the mode of DNA packaging in the sperm nucleus. The most important of these is that the DNA in the mouse sperm nucleus cannot be packaged in nucleosomes. The protamines in sperm chromatin do not function as structural proteins, providing a subunit core around which the DNA is wrapped, but appear to completely neutralize the phosphodiester backbone of the DNA molecule, thereby minimizing the repulsion between neighboring segments of DNA and allowing it to be condensed into a biochemically inactive particle of genetic information.     

HIV replication in CD4+ T lymphocytes in the presence and absence of follicular dendritic cells: inhibition of replication mediated by α-1-antitrypsin through altered IκBα ubiquitination

Follicular dendritic cells (FDCs) increase HIV replication and virus production in lymphocytes by increasing the activation of NF-κB in infected cells. Because α-1-antitrypsin (AAT) decreases HIV replication in PBMCs and monocytic cells and decreases NF-κB activity, we postulated that AAT might also block FDC-mediated HIV replication. Primary CD4(+) T cells were infected with HIV and cultured with FDCs or their supernatant with or without AAT, and ensuing viral RNA and p24 production were monitored. NF-κB activation in the infected cells was also assessed. Virus production was increased in the presence of FDC supernatant, but the addition of AAT at concentrations >0.5 mg/ml inhibited virus replication. AAT blocked the nuclear translocation of NF-κB p50/p65 despite an unexpected elevation in associated phosphorylated and ubiquitinated IκBα (Ub-IκBα). In the presence of AAT, degradation of cytoplasmic IκBα was dramatically inhibited compared with control cultures. AAT did not inhibit the proteasome; however, it altered the pattern of ubiquitination of IκBα. AAT decreased IκBα polyubiquitination linked through ubiquitin lysine residue 48 and increased ubiquitination linked through lysine residue 63. Moreover, lysine reside 63-linked Ub-IκBα degradation was substantially slower than lysine residue 48-linked Ub-IκBα in the presence of AAT, correlating altered ubiquitination with a prolonged IκBα t(1/2). Because AAT is naturally occurring and available clinically, examination of its use as an inhibitory agent in HIV-infected subjects may be informative and lead to the development of similar agents that inhibit HIV replication using a novel mechanism.     

Effects of serum type on growth and permeability properties of cultured endothelial cells

Serum is frequently added to defined basal media as a source of certain nutrients and macromolecular growth factors essential for cell growth. The many different sera commercially available may not be equally suitable for all cell types. The effects of four sera, fetal bovine serum (FBS), calf bovine serum (CS), equine serum (ES-1), and plasma-derived equine serum (ES-2), on growth and permeability properties of cultured porcine endothelial cells were determined. The rate of DNA synthesis, measured as [3H]thymidine incorporation, reached a peak at around 24 h, regardless of serum type, and was most marked with ES-1- or ES-2-treated cells. However, when estimated by total DNA, FBS, CS, or ES-1 treatment resulted in greater cell proliferation than ES-2. Based on protein synthetic rate and total cell protein, both FBS and CS appeared to be most growth supporting. At 72 h after cell plating, albumin passage across cultured endothelial monolayers was elevated in ES-1- and ES-2-treated cells compared with FBS- or CS-treated cells. "Leaky" cell monolayers were most marked with ES-1-treated cells. Cells grown in ES-2- and particularly in ES-1-enriched media were larger and more spindle-shaped compared with the typical cobblestone appearance of cells cultured in media enriched with either FBS or CS. These data suggest that CS, but not ES-1 or ES-2, is an excellent substitute for FBS to support desirable growth properties of macrovascular endothelial cells in culture.