Aberrant Types Research Articles

Gene expression analysis reveals marked differences in the transcriptome of infantile hemangioma endothelial cells compared to normal dermal microvascular endothelial cells

BackgroundInfantile hemangiomas are benign vascular tumors primarily found on the skin in 10% of the pediatric population. The etiology of this disease is largely unknown and while large scale genomic studies have examined the transcriptomes of infantile hemangioma tumors as a whole, no study to date has compared the global gene expression profiles of pure infantile hemangioma endothelial cells (HEMECs) to that of normal human dermal microvascular endothelial cells (HDMVECs).MethodsTo shed light on the molecular differences between these normal and aberrant dermal endothelial cell types, we performed whole genome microarray analysis on purified cultures of HEMECs and HDMVECs. We then utilized qPCR and immunohistochemistry to confirm our microarray results.ResultsOur array analysis identified 125 genes whose expression was upregulated and 104 genes whose expression was downregulated by greater than two fold in HEMECs compared to HDMVECs. Bioinformatics analysis revealed three major classifications of gene functions that were altered in HEMECs including cell adhesion, cell cycle, and arachidonic acid production. Several of these genes have been reported to be critical regulators and/or mutated in cancer, vascular tumors, and vascular malformations. We confirmed the expression of a subset of these differentially expressed genes (ANGPT2, ANTXR1, SMARCE1, RGS5, CTAG2, LTBP2, CLDN11, and KISS1) using qPCR and utilized immunohistochemistry on a panel of paraffin embedded infantile hemangioma tumor tissues to demonstrate that the cancer/testis antigen CTAG2 is highly abundant in vessel-dense proliferating infantile hemangiomas and with significantly reduced levels during tumor involution as vascular density decreases.ConclusionOur data reveal that the transcriptome of HEMECs is reflective of a pro-proliferative cell type with altered adhesive characteristics. Moveover, HEMECs show altered expression of many genes that are important in the progression and prognosis of metastatic cancers.

Quantitation of Spatially-Localized Proteins in Tissue Samples using MALDI-MRM Imaging

MALDI imaging allows the creation of a "molecular image" of a tissue slice. This image is reconstructed from the ion abundances in spectra obtained while rastering the laser over the tissue. These images can then be correlated with tissue histology to detect potential biomarkers of, for example, aberrant cell types. MALDI, however, is known to have problems with ion suppression, making it difficult to correlate measured ion abundance with concentration. It would be advantageous to have a method which could provide more accurate protein concentration measurements, particularly for screening applications or for precise comparisons between samples. In this paper, we report the development of a novel MALDI imaging method for the localization and accurate quantitation of proteins in tissues. This method involves optimization of in situ tryptic digestion, followed by reproducible and uniform deposition of an isotopically labeled standard peptide from a target protein onto the tissue, using an aerosol-generating device. Data is acquired by MALDI multiple reaction monitoring (MRM) mass spectrometry (MS), and accurate peptide quantitation is determined from the ratio of MRM transitions for the endogenous unlabeled proteolytic peptides to the corresponding transitions from the applied isotopically labeled standard peptides. In a parallel experiment, the quantity of the labeled peptide applied to the tissue was determined using a standard curve generated from MALDI time-of-flight (TOF) MS data. This external calibration curve was then used to determine the quantity of endogenous peptide in a given area. All standard curves generate by this method had coefficients of determination greater than 0.97. These proof-of-concept experiments using MALDI MRM-based imaging show the feasibility for the precise and accurate quantitation of tissue protein concentrations over 2 orders of magnitude, while maintaining the spatial localization information for the proteins.

Treatment of MCL Cells with Combined Rituximab and Lenalidomide Enhances NK-Mediated Synapse Formation and Cell Killing.

Abstract 1687Poster Board I-713 IntroductionMantle cell lymphoma (MCL) is a subtype of aggressive B-cell non-Hodgkin's Lymphoma (NHL) characterized by poor prognosis and very few therapeutic options that improve survival. Lenalidomide is an immunomodulatory agent that has demonstrated significant clinical activity in the treatment of patients with MCL. A mechanism attributed to lenalidomide, which has also been demonstrated in B-CLL models, is the restoration of impaired T-cell activity to form immunological synapses, thus enhancing T-cell effector functions thereby eliminating aberrant tumor B cells. Natural killer (NK) cells are another cell type that eliminates aberrant cell types via a mechanism dependent on active formation of immunological synapses. By engaging in both direct killing and ADCC, NK cells are an important component of innate immunity eliminating transformed cell types. Here we assess the effect of lenalidomide on the ability of NK cells to form immune synapses with MCL cell lines and cell samples from MCL patients. We also evaluate whether lenalidomide-mediated immunologic activity is altered when lenalidomide is combined with rituximab, an anti-CD20 antibody shown to eliminate MCL cells through the ADCC mechanism. MethodsTo measure immune synapse formation, NK and JeKo-1 cells were pre-treated with DMSO or 1μM lenalidomide for 24 or 48 hrs. JeKo-1 cells (labeled with red PKH26) were incubated for 30 min with or without 10 μg/ml rituximab, and cell conjugates were fixed and stained with Phalloidin-FITC to measure mean fluorescent intensity and F-actin formation. Adenylate kinase (AK) release and 7AAD staining were used to measure NK-mediated cytotoxicity after NK and MCL cells were co-cultured at different target-to-effector ratios, and with and without rituximab. Flow cytometry was used to measure relative expression of cell surface markers CD56 (N-CAM), CD54 (I-CAM1), and NKG2D in both JeKo-1 cells and in B-cell samples from MCL patients. ResultsAddition of lenalidomide enhanced the formation of immunological synapses between JeKo-1 cells and NK cells, increasing the number of synapses approximately 3-fold after 24 hrs of treatment. When lenalidomide was combined with rituximab the number of synapses increased 3.5-fold. A significant increase in the number of synapses also occurred when NK cells co-cultured with MCL patient samples were treated with lenalidomide (2.5-fold increase) and lenalidomide plus rituximab (3-fold increase). Lenalidomide also augmented the intensity of F-actin at the synaptic site, which indicates more matured synapses rich with F-actin. The increased synapse-forming activity resulting from treatment with rituximab plus lenalidomide also translated into enhanced NK-mediated cytotoxicity. After 48 hours of concurrent treatment with lenalidomide and rituximab, approximately 80% of JeKo-1 cells released AK compared with approximately 45% after treatment with lenalidomide alone. Comparable cell killing data upon treatment with lenalidomide plus rituximab was obtained using a 7AAD assay. Finally, to understand the mechanism by which lenalidomide enhances the formation of immune synapses and augments the rituximab-dependent NK cell-mediated cytotoxicity in MCL cells, we studied the effect of lenalidomide on effector and target cells separately. In the NK cells, lenalidomide treatment induced F-actin polymerization and polarization, and the accumulation of perforin, which is evidence for effector cell activation. Previously we showed that lenalidomide treatment of tumor cells induces changes in actin structure and increases expression of cell surface markers, including CD54 and co-stimulatory molecules, which correlated with increased antigen presentation properties of tumor cells. Here we show that lenalidomide and rituximab induced F-actin polymerization and polarization in the JeKo-1 cells when administered individually and in combination, and resulted in slightly increased cytotoxicity. ConclusionOur results suggest that in MCL, combined use of lenalidomide and rituximab enhances NK-mediated immune synapse formation and the resultant cytotoxicity. Combining lenalidomide with rituximab may also enhance the anti-tumor immune response mediated through enhanced activity of NK cells. These studies suggest that lenalidomide plus rituximab may have clinical utility in the treatment of patients with MCL. DisclosuresGaidarova:Celgene: Employment, Equity Ownership. Corral:Celgene: Employment. Glezer:Celgene: Employment, Equity Ownership. Schafer:Celgene: Employment. Lopez-Girona:Celgene: Employment.

Dose response relationships for chromosome aberrations induced by low doses of alpha-particle radiation

Using a single colour fluorescence in situ hybridisation technique, dose-responses were derived for a range of chromosomally aberrant cell types and categories of aberrations induced in peripheral blood lymphocytes by alpha-particle radiation and analysed in their first in vitro division. For a range of doses that resulted predominantly in targeted cells receiving a single hit, i.e. 0-200 mGy, linear models fitted all the different categories of aberrant cells and aberration types but the profile of chromosome damage differed for 500 mGy, reflecting the effect of different track structure.

Stability in Distributions of Black-Capped, Carolina, and Aberrant Chickadee Song Types in Illinois

Chickadee songs were recorded in 1997–2000 at 185 sites in 15 Illinois counties to map the distributions of song types of Black-capped (Poecile atricapillus) and Carolina chickadees (P. carolinensis). These species are parapatrically distributed from New Jersey to Kansas. Hybrid zones form in areas of sympatry and chickadees in these areas frequently exhibit unusual (i.e., aberrant) singing behaviors. The presence of aberrant vocalizations and/or the co-occurrence of the two species' song types at a site or by a single individual were considered evidence of a hybrid zone. Four hybrid zones were detected in Illinois along the range interface of these species. A comparison of the distributions of Black-capped, Carolina, and aberrant song types from this and a previous study conducted in Illinois in 1954–59 revealed little change has occurred in the distributions of these two species' song types. This overall stability of song type distributions indicates the northward movement of hybrid zones seen at other locations has probably not occurred in Illinois.

Effect of coat color mutations on behavioral polymorphism in farm populations of American minks (Mustela vison Schreber, 1777) and sables (Martes zibellina Linnaeus, 1758)

Behavioral polymorphism estimated by the expression of the defensive reaction towards humans has been studied in farm-bred American minks and sables with different color types. Most animals (both minks and sables) from farm populations displayed passive defensive behavior towards humans in the standard hand catch test. Coat color genes have been found to have pleiotropic effects; they influence both the penetrance and expressivity of domestication behavior: in animals with aberrant color types (both sapphire minks and white-and-black sables), the proportion of animals with domestication behavior and the expressivity of this behavior are significantly higher (p <0.01 and p < 0.001, respectively).

Topics in Sem Anesth

Improving nutritional fruit quality and impacts important agro-traits such as biotic or abiotic stresses are extremely important for human civilization. Our previous study reported that manipulation of SlMX1 gene enhanced carotenoids accumulation and drought resistance in tomato. Here, RNA-Seq analysis proved to be a very useful tool to provide insights into the regulatory mechanisms of SlMX1 involved in stress resistance and enhanced secondary metabolites. Physiological analysis showed that over-expression of SlMX1 results in substantially increased broad-spectrum tolerance to a wide-range of abiotic and biotic (fungus, bacteria, virus and insects) stresses in tomato. This research appears to be of remarkable interest because enhanced terpenoids content has been achieved by increasing trichome density. In addition, we reported two types of trichome which seems to be aberrant types in tomato. This study unravels the mechanism of regulation of SlMX1, which simultaneously modulates resistance and metabolic processes through regulating key structural and regulatory genes of the corresponding pathways.

Inhibitors of Leishmania mexicana CRK3 cyclin-dependent kinase: chemical library screen and antileishmanial activity.

The CRK3 cyclin-dependent kinase of Leishmania has been shown by genetic manipulation of the parasite to be essential for proliferation. We present data which demonstrate that chemical inhibition of CRK3 impairs the parasite's viability within macrophages, thus further validating CRK3 as a potential drug target. A microtiter plate-based histone H1 kinase assay was developed to screen CRK3 against a chemical library enriched for protein kinase inhibitors. Twenty-seven potent CRK3 inhibitors were discovered and screened against Leishmania donovani amastigotes in vitro. Sixteen of the CRK3 inhibitors displayed antileishmanial activity, with a 50% effective dose (ED50) of less than 10 microM. These compounds fell into four chemical classes: the 2,6,9-trisubstituted purines, including the C-2-alkynylated purines; the indirubins; the paullones; and derivatives of the nonspecific kinase inhibitor staurosporine. The paullones and staurosporine derivatives were toxic to macrophages. The 2,6,9-trisubstituted purines inhibited CRK3 in vitro, with 50% inhibitory concentrations ranging from high nanomolar to low micromolar concentrations. The most potent inhibitors of CRK3 (compounds 98/516 and 97/344) belonged to the indirubin class; the 50% inhibitory concentrations for these inhibitors were 16 and 47 nM, respectively, and the ED50s for these inhibitors were 5.8 and 7.6 microM, respectively. In culture, the indirubins caused growth arrest, a change in DNA content, and aberrant cell types, all consistent with the intracellular inhibition of a cyclin-dependent kinase and disruption of cell cycle control. Thus, use of chemical inhibitors supports genetic studies to confirm CRK3 as a validated drug target in Leishmania and provides pharmacophores for further drug development.

Human Fibroblasts with Mutations in COL5A1 and COL3A1 Genes Do Not Organize Collagens and Fibronectin in the Extracellular Matrix, Down-regulate α2β1 Integrin, and Recruit αvβ3 Instead of α5β1 Integrin

Dermal fibroblasts derived from types I and IV Ehlers-Danlos syndrome (EDS) patients, carrying mutations in COL5A1 and COL3A1 genes, respectively, synthesize aberrant types V and III collagen (COLL) and show defective organization of these proteins into the extracellular matrix (ECM) and high reduction of their functional receptor, the alpha(2)beta(1) integrin, compared with control fibroblasts. EDS cells also show reduced levels of fibronectin (FN) in the culture medium and lack an FN fibrillar network. Finally, EDS cells prevalently organize alpha(v)beta(3) integrin instead of alpha(5)beta(1) integrin. The alpha(v)beta(3) integrin, distributed on the whole EDS cell surface, shows FN binding and assembly properties when the cells are treated with purified FN. Treatment of EDS cells with purified COLLV or COLLIII, but not with FN, restores the control phenotype (COLL(+), FN(+), alpha(v)beta(3)(-), alpha(5)beta(1)(+), alpha(2)beta(1)(+)). Function-blocking antibodies to COLLV, COLLIII, or alpha(2)beta(1) integrin induce in control fibroblasts an EDS-like phenotype (COLL(-), FN(-), alpha(v)beta(3)(+), alpha(5)beta(1)(-), alpha(2)beta(1)(-)). These results show that in human fibroblasts alpha(2)beta(1) integrin organization and function are controlled by its ligand, and that the alpha(2)beta(1)-COLL interaction, in turn, regulates FN integrin receptor recruitment: high alpha(2)beta(1) integrin levels induce alpha(5)beta(1) integrin organization, while low alpha(2)beta(1) integrin levels lead to alpha(v)beta(3) integrin organization.

A note on aberrant pelage colors in a wild population of the gray red-backed vole Clethrionomys rufocanus bedfordiae in Hokkaido

The pelage of adult gray red-backed voles Clethrionomys rufocanus bedfordiae (Rodentia, Arvicolinae) in Hokkaido is usually red-brown or black on the dorsum and ivory or gray-ivory on the side-venter (Fig. 1a; Ota 1984; Nakata et al. 1989). However, pelage aberrations have been observed on several occasions in natural populations of C. rufocanus bedfordiae (Aizawa and Makino 1938; Fujimaki 1974; Murakawa 1986; Miyatsu et al. 1988; Takahashi 1988; Nakata et al. 1989). Although pelage color aberrations of this vole have been known of since 1938, there have been no records of several individuals with different pelage color aberrations being captured at one time from a single locality (Nakata et al. 1989). In July 2003, however, three specimens were collected from a locality in Hokkaido, these three were aberrant types with light brown buff pelage and partially white pelage. Ecological and genetic perspectives on these aberrations are expressed.

Extension of a Pulmonary Blastoma Into the Left Atrium

Extension of a Pulmonary Blastoma Into the Left Atrium

Cellular effects of leishmanial tubulin inhibitors on L. donovani

To aid our investigation of tubulin as an antileishmanial drug target, the effects of the mammalian antimicrotubule agents ansamitocin P3, taxol, and hemiasterlin on Leishmania donovani promastigotes were described. These drugs affected the assembly of purified leishmanial tubulin and inhibited the growth of L. donovani promastigotes at micromolar concentrations. When promastigotes were treated with these agents, mitotic partitioning of nuclear DNA and cytokinesis were usually inhibited. The spatial orientation of kinetoplasts was often disturbed, suggesting a role for microtubules in the segregation of these organelles during mitosis. Aberrant cell types produced in drug-treated cultures included parasites with one nucleus and two geometrically distinct kinetoplasts, parasites with multiple kinetoplasts, and cytoplasts containing a kinetoplast but no nucleus. A subset of unique cell types, parasites containing two nuclei, a spindle fiber, and two geometrically distinct kinetoplasts, were observed in hemiasterlin-treated cultures. Flow cytometric analysis of L. donovani promastigotes treated with these three drugs indicated a dramatic shift toward the G 2+M phase of the cell cycle, with some cells containing four times the amount of DNA present in G 1. These results were used to evaluate the cellular effects of WR85915, an aromatic thiocyanate with in vitro antileishmanial and anti-tubulin activity, on L. donovani. Treatment of parasites with WR85915 did not produce the unusual cell types described above and did not cause the accumulation of parasites in G 2+M, suggesting that WR85915 acts on target(s) in Leishmania in addition to tubulin. These studies validate tubulin as a suitable antileishmanial drug target and provide criteria to assess the cellular mechanism of action of new candidate antileishmanial agents.

Molecular analysis of new subtypes of ELE/RET rearrangements, their reciprocal transcripts and breakpoints in papillary thyroid carcinomas of children after Chernobyl.

A high prevalence of RET rearrangements is found in papillary thyroid carcinomas (PTC) of children from Belarus after the Chernobyl reactor accident. The ELE/RET rearrangement (PTC3) is prevailing. Aberrant types of ELE/RET rearrangement have been found with a truncated ELE1 gene: As compared with the common form (PTC3r1) one aberrant type is shorter by one 144 bp exon (PTC3r2) (three cases); in the second atypic form (PTC3r3) the ELE1 part is 18 bp shorter than in PTC3r1. In agreement with the observation that the oncogenic RET is generated by a paracentric inversion at chromosome 10, we found not only ELE/RET, but also RET/ELE transcripts in these tumors. Sequencing of the breakpoint regions at the genomic DNA level revealed DNA modifications that might be relevant for illegitimate recombination after DNA doublestrand breaks. The high prevalence of ELE/RET rearrangements and various subtypes appears to be typical for radiation-induced thyroid carcinomas of children after the Chernobyl reactor accident.

Heterochromatin variation in chromosome X in a natural population of Anopheles willmori (Diptera: Culicidae) of Thailand.

Among the eight families of Anopheles willmori derived from individual wild-caught females collected from Chiangmai Province (northern Thailand) and examined, four isofemale lines showed variation in the X chromosome, including the normal X1 and three aberrant types (X3, X4 and XL). It is postulated that these different types of X chromosomes could have arisen as a result of spontaneous chromosomal rearrangements involving tandem translocation and paracentric inversion followed by acquisition of constitutive heterochromatin. Such rare events of chromosomal changes have become established in the natural population of An. willmori in northern Thailand.

Regeneration of intergeneric somatic hybrid plants between Atropa belladonna L. and Hyoscyamus muticus L.

In the present experiments, cell suspension derived protoplast of Atropa belladonna L. (2 n = 72) var acuminata and protoplasts of an achlorphyllous mutant cell line of Egyptian henbane, Hyoscyamus muticus (2 n = 28) were fused to obtain intergeneric somatic hybrid plants. The somatic hybrid plants possessed variable chromosome complements. Those plants with a chromosome number of around 100 and with both parental genomes distinguished on the basis of chromosome size were easier to establish in the glasshouse. Plants with multiple complements (chromosome number = 178) were grossly aberrant types and very difficult to maintain. The work involved complementation at the intergeneric level within the same tribe and evidence of chromosome elimination of Atropa chromosomes was obtained. The somatic hybrid plants developed with intermediate types of leaves and altered characteristics reflecting expression of both the genomes. In one of the somatic hybrid clones, SH-15, the pentamerous orientation of the whorls of sepals, petals and anthers in both the parents was altered to a hexamerous form. Further, the parents possess a bilocular ovary and this was altered to a trilocular form in this somatic hybrid. Plants that have responded better in their development in the glasshouse had a near tetraploid chromosome number of H. muticus and a near haploid chromosome number of A. belladonna. The presence of complementary bands of esterases and peroxidases as studied in the line SH-15, affirmed the hybrid nature.

Kinetics of carrot somatic embryo development in suspension culture

Somatic embryos in liquid culture can serve as a mass cloning system in a plant propagation program. A quantitative formulation of embryo development obtained from cell suspension cultures is used to develop a segregated kinetic model. The model is based on standard classification schemes as previously developed by plant physiologists. Dependent variables include carbohydrate concentrations (sucrose, fructose, and glucose) and biomass apportioned among the inoculum (free single cells, cell clusters), normal developmental stages, and aberrant cell and embryo types. Good agreement between the model and experimental results is indicated and allows for a rigorous approach to media optimization and reactor scaleup for embryo formation.

Halteria bifurcataTamar, 1968, distribution and variation, and Halteria grandinellaO. F. Müller, 1773

Summary Waters in large parts of the United States were sampled for Halteria bifurcata and Halteria grandinella. Live specimens were examined and measured by dark phase-contrast microscopy and photomicrography. The two species were mostly found together. H. bifurcata, for which measurements are provided, normally has a barb on one of its bristles number 1. Two new aberrant forms of H. bifurcata, as well as the previously reported aberrant types, are described and discussed. All the aberrant H. bifurcata forms are associated with shallow waters. Dimensions for several H. grandinella stocks are given and effects of ambient temperatures on structure are suggested.

Method to Evaluate Pollen Viability of Upland Cotton: Tests with Chromosome Translocations

Cytogenetically induced semisterility can provide a useful phenotype to facilitate many sorts of genetic and cytogenetic manipulations that otherwise require meiotic analyses. Most chromatin deficiencies are not pollen‐transmissible in Upland cotton (Gossypium hirsutum L.) due to reduced viability. A quick and reliable technique for the assessment of cotton pollen fertility has not been established. A modified fluorochrome reaction (FCR) method, using fluorescein diacetate, was evaluated for application as a pollen viability/fertility indicator by scoring fluorescence of pollen from normal and reciprocal chromosome translocation cytotypes grown in greenhouse and field environments. Analyzed materials included 44 different true‐breeding translocation homozygotes, respective F1 heterozygotes, and three different cytogenetically analyzed BC1F1 families. Translocation heterozygotes consistently produced higher frequencies (12.3–38.8%) of less intensely fluorescing pollen grains than homozygotes (0.4–6.4%) or normals (0.4–4.0%). Results were concordant with cytogenetic expectations and meiotic analyses, indicating that these and perhaps other cytogenetically aberrant types of cotton can be discerned phenotypically on the basis of percent pollen viability, thereby facilitating their detection and use as cytogenetic tools.

Streptokinase-dependent delayed activation of horse plasminogen

Complete activation of purified horse plasminogen to plasmin was obtained with a 1:10 molar ratio of streptokinase to plasminogen after 5 min of incubation at 37°C. At a 1:1 molar ratio, maximal activity did not appear until 15–30 min, while at a ratio of 6:1 complete activation was delayed for 120–180 min. Gel filtration studies of isotopically labeled streptokinase and horse plasminogen suggest that the delay was due to impaired formation of a streptokinase-plasminogen complex. The predominant streptokinase moiety within the streptokinase-plasmin complex which forms from the streptokinase-plasminogen complex had a molecular weight of about 25000. The streptokinase-horse plasmin complex activated bovine plasminogen and was relatively stable. Native streptokinase was rapidly modified by horse plasmin predominantly to a fragment with a molecular weight comparable to that of the streptokinase moiety within the horse streptokinase-plasmin complex, about 25000 daltons. Partial characterization of horse plasminogen revealed no striking differences from human plasminogen in terms of molecular weight, N-terminal analysis and amino acid composition. However, horse plasminogen did not react with antibodies to human plasminogen, and its isoenzymes were more acidic than those of the human. Further characterization of horse plasminogen will be required to ascertain whether activation by streptokinase can serve as a model for the altered kinetics which have recently been described for the activation of aberrant types of human plasminogen.

Plumbate Origins Revisited

Plumbate, a distinctive ceramic of the Early Postclassic, has been thought to have been manufactured in the Pacific region of Guatemala and traded throughout Mesoamerica and Central America. Two aberrant types of Plumbate found in Late Classic contexts in Veracruz and Early Postclassic contexts in central El Salvador suggest that the origins and spread of Plumbate or Plumbate techniques may be more complex.