Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

The Role of Historical Barriers in the Diversification Processes in Open Vegetation Formations during the Miocene/Pliocene Using an Ancient Rodent Lineage as a Model

The Neotropics harbors a high diversity of species and several hypotheses have been proposed to account for this pattern. However, while species of forested domains are frequently studied, less is known of species from open vegetation formations occupying, altogether, a larger area than the Amazon Forest. Here we evaluate the role of historical barriers and the riverine hypothesis in the speciation patterns of small mammals by analyzing an ancient rodent lineage (Thrichomys, Hystricomorpha). Phylogenetic and biogeographic analyses were carried out with mitochondrial and nuclear DNA markers to analyze the evolutionary relationships between Thrichomys lineages occurring in dry domains along both banks of the Rio São Francisco. This river is one of the longest of South America whose course and water flow have been modified by inland tectonic activities and climate changes. Molecular data showed a higher number of lineages than previously described. The T. inermis species complex with 2n = 26, FN = 48 was observed in both banks of the river showing a paraphyletic arrangement, suggesting that river crossing had occurred, from east to west. A similar pattern was also observed for the T. apereoides complex. Thrichomys speciation occurred in Late Miocene when the river followed a different course. The current geographic distribution of Thrichomys species and their phylogenetic relationships suggested the existence of frequent past connections between both banks in the middle section of the Rio São Francisco. The extensive palaeodune region found in this area has been identified as a centre of endemism of several vertebrate species and is likely to be a center of Thrichomys diversification.     

Identification of Bacterial Infection in Neotropical Primates

Emerging infectious diseases usually arise from wild animal populations. In the present work, we performed a screening for bacterial infection in natural populations of New World primates. The blood cell bulk DNAs from 181 individuals of four Platyrrhini genera were PCR screened for eubacterial 16S rRNA genes. Bacteria were detected and identified in 13 distinct individuals of Alouatta belzebul, Alouatta caraya, and Cebus apella monkeys from geographically distant regions in the states of Mato Grosso and Pará, Brazil. Sequence analyses showed that these Platyrrhini bacteria are closely related not only to human pathogens Pseudomonas spp. but also to Pseudomonas simiae and sheep-Acari infecting Pseudomonas spp. The identified Pseudomonas possibly represents a group of bacteria circulating in natural monkey populations.     

Molecular phylogenetics of howler monkeys (Alouatta, Platyrrhini). A comparison with karyotypic data

Molecular phylogenetic analyses of seven Brazilian Alouatta species, based on cytochrome b DNA sequence data were carried out. Parsimony and neighbor joining topologies grouped Alouatta belzebul and A. fusca as sister groups in one clade while another, well-supported clade contained A. seniculus as the most basal offshoot, followed by A. nigerrima as a sister lineage of A. macconnelli/A. stramineus. Estimates of inter-specific sequence divergence were generally low, and estimates of the time of divergence indicated that the main Alouatta lineages emerged during a short evolutionary interval. A comparison with karyotypic data confirmed the molecular topology showing a closer relationship between A. macconnelli and A. stramineus in respect to A. nigerrima. It also showed that the XX/XY sex chromosome system was maintained in several lineages while the X1X2Y/X1X1X2X2 system appeared independently at least three times during the radiation of howler monkeys. Moreover, the X1X2Y1Y2/X1X1X2X2 system might have appeared once or, alternatively, twice and independently.     

Morphologic, karyotypic, and molecular evidence of a new form of Chiropotes (primates, pitheciinae)

Morphologic, karyotypic, and molecular analyses were carried out in 25 specimens of a distinct morph of Chiropotes (henceforth termed Chiropotes sp.) obtained from a number of localities in the Brazilian Amazon. Pelage coloration clearly distinguishes the collected specimens and all other known species of this genus. A distinct karyotype was described for Chiropotes sp. It differs from C. satanas chiropotes by two pericentric inversions, and from C. satanas utahicki by three, which suggests that these taxa are reproductively isolated. Morphometric analyses did not show significant differentiation between these Chiropotes taxa. Molecular analyses confirmed the monophyly of the subfamily Pitheciinae and genera Chiropotes, Cacajao, and Pithecia (the latter appearing as the most basal lineage of the pithecine clade). The genetic distances between C. s. utahicki and Chiropotes sp. from Rio Negro were greater than those between three recognized species of Pithecia, but smaller than those between Cacajao calvus and Cacajao melanocephalus. The most appropriate name for Chiropotes sp. from Rio Negro is C. israelita. This species, C. s. chiropotes, and C. s. utahicki are allopatric. Pelage coloration, karyotype, and molecular analysis strongly indicate that C. chiropotes, C. utahicki, and Chiropotes israelita deserve species status.     

Dietary input of microbes and host genetic variation shape among-population differences in stickleback gut microbiota

To explain differences in gut microbial communities we must determine how processes regulating microbial community assembly (colonization, persistence) differ among hosts and affect microbiota composition. We surveyed the gut microbiota of threespine stickleback (Gasterosteus aculeatus) from 10 geographically clustered populations and sequenced environmental samples to track potential colonizing microbes and quantify the effects of host environment and genotype. Gut microbiota composition and diversity varied among populations. These among-population differences were associated with multiple covarying ecological variables: habitat type (lake, stream, estuary), lake geomorphology and food- (but not water-) associated microbiota. Fish genotype also covaried with gut microbiota composition; more genetically divergent populations exhibited more divergent gut microbiota. Our results suggest that population level differences in stickleback gut microbiota may depend more on internal sorting processes (host genotype) than on colonization processes (transient environmental effects).     

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

The primates of the Neotropics: genomes and chromosomes

The classification of neotropical primates has been controversial. Different arrangements have been proposed, depending on taxonomic criteria and on the traits selected for phylogenetic reconstructions. These include gross morphologic characters, karyotypic attributes and DNA sequence data of nuclear and mitochondrial genes and of repetitive genomic components. These approaches have substantially clarified the main intergeneric relationships although several intrageneric arrangements still remain to be elucidated. In this review, we compare karyologic and molecular data of this speciose group.     

Karyologic diversification and phylogenetic relationships of the genus<Emphasis Type="Italic">Thalpomys</Emphasis> (Rodentia,Sigmodontinae)

We describe the karyotype ofThalpomys species, from different Brazilian localities of the Cerrado.Thalpomys cerradensis Herskovitz, 1990 showed 2n=36, FN=34 andT. lasiotis Thomas, 1916 2n=38, FN=38. Comparisons of G-band karyotypes showed evident inter-specific homologies indicating that their chromosome complements could be derived from one another by two presumed rearrangements. Both species showed pericentromeric C-band regions in almost all chromosomes but a comparison with CMA3/DA/DAPI staining indicated that the molecular content of heterochromatic regions was different.T. lasiotis specimens from two different localities differed in the morphology of the X chromosome due to the presence of a short heterochromatic arm. These chromosome types are apparently fixed in each population rather than maintained as a polymorphic variation. Phylogenetic analyses supported the monophyly of the genusThalpomys but was not capable of elucidating its phylogenetic relationship to other Akodontini rodents. These analyses also showed inter-individual variation inT. lasiotis, even within a given population. Phylogenetic analyses placedT. lasiotis specimens with different karyotypes in different monophyletic branches. Molecular and karyologic data confirmed the identity of the genusThalpomys.