Reovirus serotype 1/strain Lang-stimulated activation of antigen-specific T lymphocytes in Peyer's patches and distal gut-mucosal sites: activation status and cytotoxic mechanisms

Intraduodenal priming of mice with reovirus serotype 1/strain Lang (reovirus 1/L) stimulates gut lymphocytes and generates precursor and effector CTLs. Our earlier studies demonstrated that germinal center and T cell Ag (GCT) is a marker which identifies reovirus 1/L-specific precursor CTL and effector CTL in Peyer's patches (PP) of reovirus 1/L-inoculated mice. In this study, we characterized the expression of the activation markers, GCT and CD11c, on reovirus 1/L-stimulated gut lymphocytes and the effector mechanisms involved in reovirus 1/L-specific cytotoxicity. We found that intraduodenal reovirus 1/L inoculation of mice induced the expression of both GCT and CD11c on PP lymphocytes (PPL), intraepithelial lymphocytes (IEL), and lamina propria lymphocytes (LPL), and these activated cells expressed Fas ligand (FasL). The majority of the GCT+ CD11c+ IEL and LPL expressed a phenotype, TCRalphabeta+ Thy-1+ CD8+ similar to that expressed on reovirus 1/L-stimulated PPL. However, splenic lymphocytes expressed GCT but not CD11c after stimulation with reovirus 1/L. Perforin, Fas-FasL, and TRAIL pathways were found to be involved in PPL, IEL, and LPL cytotoxic activity against reovirus 1/L-infected targets. In PPL, perforin and Fas-FasL pathways were more effective than TRAIL. In IEL, all three cytotoxic mechanisms were equally as effective. However, LPL prefer Fas-FasL and TRAIL over perforin. Further, we demonstrated the preferential migration of GCT+ PPL to the intraepithelial compartment and the lamina propria. These results suggest that GCT and CD11c can be used as activation markers for gut lymphocytes and CD11c can also be used to differentiate between activated gut and systemic lymphocytes.     

The value of the IUCN Red List for conservation

The IUCN Red List of Threatened Species is the most comprehensive resource detailing the global conservation status of plants and animals. The 2004 edition represents a milestone in the four-decade long history of the Red List, including the first Global Amphibian Assessment and a near doubling in assessed species since 2000. Moreover, the Red List assessment process itself has developed substantially over the past decade, extending the value of the Red List far beyond the assignation of threat status. We highlight here how the Red List, in conjunction with the comprehensive data compiled to support it and in spite of several important limitations, has become an increasingly powerful tool for conservation planning, management, monitoring and decision making.     

The distribution and evolutionary history of the PRP8 intein

Background  

We recently described a mini-intein in the PRP8 gene of a strain of the basidiomycete Cryptococcus neoformans, an important fungal pathogen of humans. This was the second described intein in the nuclear genome of any eukaryote; the first nuclear encoded intein was found in the VMA gene of several saccharomycete yeasts. The evolution of eukaryote inteins is not well understood. In this report we describe additional PRP8 inteins (bringing the total of these to over 20). We compare and contrast the phylogenetic distribution and evolutionary history of the PRP8 intein and the saccharomycete VMA intein, in order to derive a broader understanding of eukaryote intein evolution. It has been suggested that eukaryote inteins undergo horizontal transfer and the present analysis explores this proposal.     

Characterization of Expressed Sequence Tag–Derived Simple Sequence Repeat Markers for Aspergillus flavus: Emphasis on Variability of Isolates from the Southern United States

Simple sequence repeat (SSR) markers were developed from Aspergillus flavus expressed sequence tag (EST) database to conduct an analysis of genetic relationships of Aspergillus isolates from numerous host species and geographical regions, but primarily from the United States. Twenty-nine primers were designed from 362 tri-nucleotide EST-SSR sequences. Eighteen polymorphic loci were used to genotype 96 Aspergillus species isolates. The number of alleles detected per locus ranged from 2 to 24 with a mean of 8.2 alleles. Haploid diversity ranged from 0.28 to 0.91. Genetic distance matrix was used to perform principal coordinates analysis (PCA) and to generate dendrograms using unweighted pair group method with arithmetic mean (UPGMA). Two principal coordinates explained more than 75?% of the total variation among the isolates. One clade was identified for A. flavus isolates (n?=?87) with the other Aspergillus species (n?=?7) using PCA, but five distinct clusters were present when the others taxa were excluded from the analysis. Six groups were noted when the EST-SSR data were compared using UPGMA. However, the latter PCA or UPGMA comparison resulted in no direct associations with host species, geographical region or aflatoxin production. Furthermore, there was no direct correlation to visible morphological features such as sclerotial types. The isolates from Mississippi Delta region, which contained the largest percentage of isolates, did not show any unusual clustering except for isolates K32, K55, and 199. Further studies of these three isolates are warranted to evaluate their pathogenicity, aflatoxin production potential, additional gene sequences (e.g., RPB2), and morphological comparisons.     

Development of isoform selective PI3-kinase inhibitors as pharmacological tools for elucidating the PI3K pathway

Using a parallel synthesis approach to target a non-conserved region of the PI3K catalytic domain a pan-PI3K inhibitor 1 was elaborated to provide alpha, delta and gamma isoform selective Class I PI3K inhibitors 21, 24, 26 and 27. The compounds had good cellular activity and were selective against protein kinases and other members of the PI3K superfamily including mTOR and DNA-PK.     

Historical and Contemporary DNA Indicate Fisher Decline and Isolation Occurred Prior to the European Settlement of California

Establishing if species contractions were the result of natural phenomena or human induced landscape changes is essential for managing natural populations. Fishers (Martes pennanti) in California occur in two geographically and genetically isolated populations in the northwestern mountains and southern Sierra Nevada. Their isolation is hypothesized to have resulted from a decline in abundance and distribution associated with European settlement in the 1800s. However, there is little evidence to establish that fisher occupied the area between the two extant populations at that time. We analyzed 10 microsatellite loci from 275 contemporary and 21 historical fisher samples (1880–1920) to evaluate the demographic history of fisher in California. We did not find any evidence of a recent (post-European) bottleneck in the northwestern population. In the southern Sierra Nevada, genetic subdivision within the population strongly influenced bottleneck tests. After accounting for genetic subdivision, we found a bottleneck signal only in the northern and central portions of the southern Sierra Nevada, indicating that the southernmost tip of these mountains may have acted as a refugium for fisher during the anthropogenic changes of the late 19^th and early 20^th centuries. Using a coalescent-based Bayesian analysis, we detected a 90% decline in effective population size and dated the time of decline to over a thousand years ago. We hypothesize that fisher distribution in California contracted to the two current population areas pre-European settlement, and that portions of the southern Sierra Nevada subsequently experienced another more recent bottleneck post-European settlement.     

UNC-45 is required for NMY-2 contractile function in early embryonic polarity establishment and germline cellularization in C. elegans

The Caenorhabditis elegans UNC-45 protein is required for proper body wall muscle assembly and acts as a molecular co-chaperone for type II myosins. In contrast to other body wall muscle components, UNC-45 is also abundant in the germline and embryo. We show that maternally provided UNC-45 acts with non-muscle myosin II (NMY-2) during embryonic polarity establishment, cytokinesis and germline cellularization. In embryos depleted for UNC-45, myosin contractility is eliminated resulting in embryonic defects in polar body extrusion, cytokinesis and establishment of polarity. Despite a lack of contractility in an unc-45(RNAi) embryo, NMY-2::GFP localizes to the cortex and accumulates at the presumptive cytokinetic furrow indicating that UNC-45 is not required for cortical localization. UNC-45 and NMY-2 are also required for fertility since the lack of either component results in complete sterility due to failed initiation of the cellularization furrows that separate syncytial nuclei into germ cells. In the absence of UNC-45, the actomyosin cytoskeleton does not contract despite non-functional myosin still directly binding actin. UNC-45 has been previously suggested to be required for the folding of the myosin head, and our results refine this hypothesis suggesting that UNC-45 is not required to fold or maintain the actin binding domain but is still required for myosin function.     

Highly polymorphic microsatellite markers developed for the social halictine bee Lasioglossum (Chilalictus) hemichalceum

Lasioglossum ( Chilalictus ) hemichalceum is a social halictine bee species for which we developed 10 polymorphic microsatellite loci in order to investigate detailed genetic structure of cooperating indvididuals. The loci are highly polymorphic with allele numbers ranging between eight and 22. A null allele was detected at one locus in the absence of pedigree information.     

A histochemical study of the regional distribution in the rat brain of enzymatic activity hydrolyzing glucose- and 2-deoxyglucose-6-phosphate

A modified Wachstein-Meisel lead salt method using glucose-6-phosphate or 2-deoxyglucose-6-phosphate as substrates was employed at the light microscopic level to map the rat brain for glucose-6-phosphatase (G-6-Pase). As has been described, most of the activity of the enzyme resided in neuronal cell bodies and dendritic stems. No differences were found between the results obtained with the two substrates. Two categories of brain structures with heavy and with moderate staining could be distinguished while the majority of brain regions contained only barely discernible neurons. Structures displaying very high enzyme activity included nuclei of cranial nerves, nuclei of the reticular formation, Purkinje cells, and some parts of the limbic system, e.g., CA 3 and CA 4 pyramidal fields of the hippocampus. It is pointed out that accurate biochemical determinations of G-6-Pase activity will critically depend on painstaking microdissection of nuclei and cell layers. The histochemical results may be pertinent to the interpretation of the 2-deoxyglucose method for assessment of regional glucose utilization rates in brain. The present observations make it unlikely that regional variations in G-6-Pase activity account for differences in uptake and retention of radioactivity from (1-14C)glucose and (14C)2-deoxyglucose reported previously by our group.     

Autoradiographic studies on glucose utilization in individual zones of the rat thymus

Wistar rats were injected intraperitoneally with 2-(14C)deoxyglucose and their thymuses were processed for thaw-mount autoradiography after 5, 10 or 35 min. Highest levels of radioactivity were demonstrated in the thymic medulla (5-fold higher than in the cortex). Scanning of autoradiograms for regional differences in grain densities indicated particularly intense glucose utilization in the cortico-medullary zone. Differences in glucose utilization between individual thymic zones seem to reflect differences in cellular composition, i.e., ratio of stroma cells to thymocytes.