Molecular diversity of native bradyrhizobia isolated from lima bean (Phaseolus lunatus L.) in Peru

The diversity of a collection of 21 bradyrhizobial isolates from Lima bean (Phaseolus lunatus L.) was assayed by molecular methods. Moderately high to high genetic diversity was revealed by multilocus enzyme electrophoresis (MLEE) analysis of seven enzyme loci and genomic fingerprints with ERIC and BOX primers. Two groups with differences in growth rate were found among the isolates and their differentiation as two divergent bradyrhizobial lineages was supported by PCR-RFLP of the rpoB gene and sequence analysis of the 16S rDNA and dnaK genes. Isolates with slow growth (SG) were identified as Bradyrhizobium yuanmingense, while extra-slow growing isolates (ESG) constitute a new lineage different from all described Bradyrhizobium species. Three distinct symbiotic genotypes were detected among Lima bean bradyrhizobia by PCR-RFLP and sequence analysis of the nifH and nodB genes. One genotype was found in the ESG lineage and two in B. yuanmingense. Another symbiotic genotype was detected in B. yuamingense isolated from Lespedeza plants. The identified bradyrhizobial lineages constitute sympatric species effectively nodulating Lima bean on the coast of Peru.     

What Hanuman langur males know about female reproductive status

In species with a high risk of infanticide, a conflict of interest exists between the sexes over the amount of paternity information that is available to males. While females are expected to keep males unaware of their reproductive status in order to confuse paternity, selection should favor those male traits that enhance the males' assessment of female status and consequently of paternity probability. In Hanuman langurs (Semnopithecus entellus), a species that is extremely vulnerable to infanticide, females have been shown to successfully conceal the exact timing of ovulation from males--perhaps because they exhibit no sexual swelling and mate during all reproductive phases, including gestation. Nevertheless, it remains unclear whether males have hitherto unrecognized information about females' reproductive condition on a broader level that could still enhance male reproductive success. We investigated male assessment of female reproductive states in a population of wild Hanuman langurs as indicated by changes in male behavior, such as rates of copulations, anogenital inspections, and consortships, in relation to different female receptive periods (pregnant, fertile-nonconceptional, and conceptional). Our data indicate that males were able to discern qualitatively distinct reproductive states. Males were more interested in fertile than pregnant females, as indicated by higher copulation rates. Based on consortships, males distinguished fertile from nonfertile phases, as well as fertile, nonconceptional receptive periods from conceptional ones. Hanuman langur males are thus not as unaware of female reproductive condition as previously thought, supporting the idea of an ongoing battle of the sexes over paternity information. However, granting some knowledge while at the same time concealing the exact day of ovulation may also reflect a pure female strategy of balancing paternity concentration with paternity confusion, which is the most likely strategy in this system with high infanticide risk and male defense of infants.     

A simple polymerase chain reaction-sequencing analysis capable of identifying multiple medically relevant filamentous fungal species

Due to the accumulating evidence that suggests that numerous unhealthy conditions in the indoor environment are the result of abnormal growth of the filamentous fungi (mold) in and on building surfaces it is necessary to accurately determine the organisms responsible for these maladies and to identify them in an accurate and timely manner. Historically, identification of filamentous fungal (mold) species has been based on morphological characteristics, both macroscopic and microscopic. These methods may often be time consuming and inaccurate, necessitating the development of identification protocols that are rapid, sensitive, and precise. To this end, we have devised a simple PAN-PCR approach which when coupled to cloning and sequencing of the clones allows for the unambiguous identification of multiple fungal organisms. Universal primers are used to amplify ribosomal DNA sequences which are then cloned and transformed into Escherichia coli. Individual clones are then sequenced and individual sequences analyzed and organisms identified. Using this method we were capable of identifying Stachybotrys chartarum, Penicillium purpurogenum, Aspergillus sydowii, and Cladosporium cladosporioides from a mixed culture. This method was found to be rapid, highly specific, easy to perform, and cost effective.     

How to induce non-polarized cells of hepatic origin to express typical hepatocyte polarity: generation of new highly polarized cell models with developed and functional bile canaliculi

Few in vitro models expressing complex hepatocyte polarity are available. We used the unpolarized rat Fao cell line to isolate the polarized WIF-B line. These complex rat-human hybrid cells form functional simple bile canaliculi. To obtain Fao-derived polarized models with a simpler chromosome content and developed bile canaliculi, we employed two approaches. Partial success was achieved with monochromosomal hybrids. As shown by the immunolocalization of apical, basolateral, and tight-junctional proteins, monochromosomal hybrid 11-3 cells were polarized. They formed simple functional bile canaliculi and transiently expressed the typical polarity of simple epithelial cells. One subclone blocked in this polarity state was isolated. A more robust approach was provided by spheroid culture, a three-dimensional system that strengthens cell-cell contacts. Transient spheroid culture induced irreversible polarization of Fao cells. This induction occurred in most spheroids (approximately 1% of the cells). From populations enriched in stably polarized cells, we generated new polarized cell models, designated Can. Can 3-1 cells formed simple functional bile canaliculi when plated at high density. Regardless of plating density, Can 9 and Can 10 cells formed long tubular branched canaliculi competent for vectorial transport of organic anions and bile acids, and involving several dozen adjacent cells. Thus, we have generated new cell models stably expressing typical hepatocyte polarity. Among these models, Can 9 and Can 10 are the first capable of forming functional, highly developed bile canaliculi similar to those formed in vivo. This work was supported by grants from the Association pour la Recherche sur le Cancer (no.6551), the Institut Curie (PIC Signalisation Cellulaire, no. 914) and the Institut National de la Santé et de la Recherche Médicale (contract PRISME 98-09).     

Cytomegalovirus encodes a positive regulator of antigen presentation

Murine cytomegalovirus encodes three regulators of antigen presentation to antiviral CD8 T cells. According to current paradigms, all three regulators are committed to the inhibition of the presentation of antigenic peptides. Whereas m152/gp40 catalyzes the retention of peptide-loaded major histocompatibility complex (MHC) class I molecules in a cis-Golgi compartment, m06/gp48 binds stably to class I molecules and directs them into the cellular cargo-sorting pathway of lysosomal degradation. Regulator m04/gp34 also binds stably to class I molecules, but unlike m152 and m06, it does not downmodulate MHC class I cell surface expression. It has entered the literature as a direct inhibitor of T-cell recognition of the MHC-peptide complex at the cell surface. In this work, we have studied the presentation of antigenic viral peptides in cells infected with a comprehensive set of mutant viruses expressing the three regulators separately as well as in all possible combinations. The results redefine m04 as a positive regulator dedicated to the facilitation of antigen presentation. When expressed alone, it did not inhibit T-cell recognition, and when expressed in the presence of m152, it restored antigen presentation by antagonizing the inhibitory function of m152. Its intrinsic positive function, however, was antagonized and even slightly overcompensated for by the negative regulator m06. In an adoptive cell transfer model, the opposing forces of the three regulators were found to govern immune surveillance in the infected host. While negative regulators, also known as immunoevasins, are common, the existence of a positive regulator is without precedent and indicates an intriguing genetic potential of this virus to influence antigen presentation.     

DNA-PKcs dependence of Artemis endonucleolytic activity, differences between hairpins and 5' or 3' overhangs

During V(D)J recombination, the RAG proteins create DNA hairpins at the V, D, or J coding ends, and the structure-specific nuclease Artemis is essential to open these hairpins prior to joining. Artemis also is an endonuclease for 5' and 3' overhangs at many DNA double strand breaks caused by ionizing radiation, and Artemis functions as part of the nonhomologous DNA end joining pathway in repairing these. All of these activities require activation of the Artemis protein by interaction with and phosphorylation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In this study, we have identified a region of the Artemis protein involved in the interaction with DNA-PKcs. Furthermore, the biochemical and functional analyses of C-terminally truncated Artemis variants indicate that the hair-pin opening and DNA overhang endonucleolytic features of Artemis are triggered by DNA-PKcs in two modes. First, autoinhibition mediated by the C-terminal tail of Artemis is relieved by phosphorylation of this tail by DNA-PKcs. Thus, C-terminally truncated Artemis derivatives imitate DNA-PKcs-activated wild type Artemis protein and exhibit intrinsic hairpin opening activity. Second, DNA-PKcs may optimally configure 5' and 3' overhang substrates for the endonucleolytic function of Artemis.     

Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling

Synaptic vesicles have been proposed to form through two mechanisms: one directly from the plasma membrane involving clathrin-dependent endocytosis and the adaptor protein AP2, and the other from an endosomal intermediate mediated by the adaptor AP3. However, the relative role of these two mechanisms in synaptic vesicle recycling has remained unclear. We now find that vesicular glutamate transporter VGLUT1 interacts directly with endophilin, a component of the clathrin-dependent endocytic machinery. In the absence of its interaction with endophilin, VGLUT1 recycles more slowly during prolonged, high-frequency stimulation. Inhibition of the AP3 pathway with brefeldin A rescues the rate of recycling, suggesting a competition between AP2 and -3 pathways, with endophilin recruiting VGLUT1 toward the faster AP2 pathway. After stimulation, however, inhibition of the AP3 pathway prevents the full recovery of VGLUT1 by endocytosis, implicating the AP3 pathway specifically in compensatory endocytosis.     

Induction of broad CD4+ and CD8+ T-cell responses and cross-neutralizing antibodies against hepatitis C virus by vaccination with Th1-adjuvanted polypeptides followed by defective alphaviral particles expressing envelope glycoproteins gpE1 and gpE2 and nonstructural proteins 3, 4, and 5

Broad, multispecific CD4⁺ and CD8⁺ T-cell responses to the hepatitis C virus (HCV), as well as virus-cross-neutralizing antibodies, are associated with recovery from acute infection and may also be associated in chronic HCV patients with a favorable response to antiviral treatment. In order to recapitulate all of these responses in an ideal vaccine regimen, we have explored the use of recombinant HCV polypeptides combined with various Th1-type adjuvants and replication-defective alphaviral particles encoding HCV proteins in various prime/boost modalities in BALB/c mice. Defective chimeric alphaviral particles derived from the Sindbis and Venezuelan equine encephalitis viruses encoding either the HCV envelope glycoprotein gpE1/gpE2 heterodimer (E1E2) or nonstructural proteins 3, 4, and 5 (NS345) elicited strong CD8⁺ T-cell responses but low CD4⁺ T helper responses to these HCV gene products. In contrast, recombinant E1E2 glycoproteins adjuvanted with MF59 containing a CpG oligonucleotide elicited strong CD4⁺ T helper responses but no CD8⁺ T-cell responses. A recombinant NS345 polyprotein also stimulated strong CD4⁺ T helper responses but no CD8⁺ T-cell responses when adjuvanted with Iscomatrix containing CpG. Optimal elicitation of broad CD4⁺ and CD8⁺ T-cell responses to E1E2 and NS345 was obtained by first priming with Th1-adjuvanted proteins and then boosting with chimeric, defective alphaviruses expressing these HCV genes. In addition, this prime/boost regimen resulted in the induction of anti-E1E2 antibodies capable of cross-neutralizing heterologous HCV isolates in vitro. This vaccine formulation and regimen may therefore be optimal in humans for protection against this highly heterogeneous global pathogen.     

Media formulation influences in vitro ectomycorrhizal synthesis on the European aspen Populus tremula L.

The effect of various media formulations on in vitro ectomycorrhizal synthesis of identified fungal strains with European aspen (Populus tremula L.) was tested in Petri dishes. Pre-grown seedlings were transferred to various nutrient media and inoculated with Paxillus involutus isolates using modified sandwich techniques. Mycorrhiza formation was evaluated macroscopically and further confirmed by microscopic examination of semi-thin sections for anatomical features of the mantle and the Hartig net. Standard media formulations did not support successful ectomycorrhiza formation because of either very poor plant survival (below 20%) or impaired fungal growth. The inclusion of micronutrients and vitamins in a Melin Norkrans (MMN)-based medium increased plant survival rate to above 60% and supported successful mycorrhizal synthesis. P. involutus isolates formed mycorrhizas with a characteristic Hartig net restricted to the epidermis. Mantle density and thickness varied depending on the isolate. In a follow-up experiment, the adapted medium supported successful ectomycorrhiza formation by various Laccaria and Hebeloma isolates. Our results show that an exogenous supply of vitamins and micronutrients in the medium was a prerequisite for successful mycorrhization of P. tremula in vitro in Petri dishes.