Annexins as cell-type-specific markers in the developing chicken chorionallantoic membrane

Between day E8 and E12 of embryonic development, the chicken chorioallantoic membrane (CAM) undergoes massive structural rearrangement enabling calcium-uptake from the eggshell to supply the growing embryo. However, the contribution of the various cell types of the chorionic epithelium including the capillary covering (CC) cells, villus cavity (VC) cells, endothelial-like cells, and basal cells to this developmental program is largely unknown. In order to obtain markers for the different cell types in the chorionic epithelium, we determined the expression patterns of various calcium-binding annexins in the developing chicken CAM. By reverse transcription/polymerase chain reaction with primers deduced from nucleotide sequences available in various databases, the presence of annexin (anx)-1, anx-2, anx-5, and anx-6 was demonstrated at days E8 and E12. Quantitative immunoblotting with novel antibodies raised against the recombinant proteins revealed that anx-1 and anx-5 were significantly up-regulated at day E12, whereas anx-2 and anx-6 expression remained almost unchanged in comparison to levels at day E8. Immunohistochemistry of paraffin-embedded sections of E12 CAM revealed anx-1 in CC cells and VC cells. Anx-2 was localized in capillaries in the chorionic epithelium and in basal cells of the allantoic epithelium, whereas anx-6 was detected in basal cells or endothelial-like cells of the chorionic epithelium and in the media of larger vessels in the mesenchyme. A 2-day exposure of the CAM to a tumor cell spheroid resulted in strong proliferation of anx-1-expressing CC cells suggesting that these cells participate in the embryonic response to experimental intervention. Thus, annexins exhibit complementary expression patterns and represent appropriate cell markers for the further characterization of CAM development and the interpretation of results obtained when using CAM as an experimental model.

Karyotypic characterization of infant embryonal rhabdomyosarcoma

Despite embryonal rhabdomyosarcoma (eRMS) representing the most frequent form of RMS, the karyotypic characterization of this tumor subtype is still incomplete. We report the karyotypic analysis of two new cases of infant-onset eRMS. Both cases had a hyperdiploid karyotype, including gain of chromosomes 2 and 8. Only one of the cases showed a structural aberration, an unbalanced rearrangement involving 4p. These cases, together with a review of the literature, suggest that a karyotypic subgroup exists in infant eRMS that is defined by hyperdiploidy (<53 chromosomes) and includes gain of chromosomes 2, 8, 11, and 17, with few or no structural aberrations. Hence, this report illustrates that distinct karyotypic subgroups may be found in eRMS, which ultimately may be shown to have prognostic relevance.

Plasma membrane-associated annexin A6 reduces Ca2+ entry by stabilizing the cortical actin cytoskeleton

The annexins are a family of Ca(2+)- and phospholipid-binding proteins, which interact with membranes upon increase of [Ca(2+)](i) or during cytoplasmic acidification. The transient nature of the membrane binding of annexins complicates the study of their influence on intracellular processes. To address the function of annexins at the plasma membrane (PM), we fused fluorescent protein-tagged annexins A6, A1, and A2 with H- and K-Ras membrane anchors. Stable PM localization of membrane-anchored annexin A6 significantly decreased the store-operated Ca(2+) entry (SOCE), but did not influence the rates of Ca(2+) extrusion. This attenuation was specific for annexin A6 because PM-anchored annexins A1 and A2 did not alter SOCE. Membrane association of annexin A6 was necessary for a measurable decrease of SOCE, because cytoplasmic annexin A6 had no effect on Ca(2+) entry as long as [Ca(2+)](i) was below the threshold of annexin A6-membrane translocation. However, when [Ca(2+)](i) reached the levels necessary for the Ca(2+)-dependent PM association of ectopically expressed wild-type annexin A6, SOCE was also inhibited. Conversely, knockdown of the endogenous annexin A6 in HEK293 cells resulted in an elevated Ca(2+) entry. Constitutive PM localization of annexin A6 caused a rearrangement and accumulation of F-actin at the PM, indicating a stabilized cortical cytoskeleton. Consistent with these findings, disruption of the actin cytoskeleton using latrunculin A abolished the inhibitory effect of PM-anchored annexin A6 on SOCE. In agreement with the inhibitory effect of annexin A6 on SOCE, constitutive PM localization of annexin A6 inhibited cell proliferation. Taken together, our results implicate annexin A6 in the actin-dependent regulation of Ca(2+) entry, with consequences for the rates of cell proliferation.

The laulimalide family: total synthesis and biological evaluation of neolaulimalide, isolaulimalide, laulimalide and a nonnatural analogue

We herein describe in full detail the first total synthesis of the antitumor agents neolaulimalide and isolaulimalide as well as a highly efficient route to laulimalide. A Kulinkovich reaction followed by a cyclopropyl-allyl rearrangement is used to install the exo-methylene group. The C(2)-C(16) aldehyde fragment is coupled with the C(17)-C(28) sulfone fragments by a highly (E)-selective Julia-Lythgoe-Kocienski olefination to deliver the key intermediates of all three syntheses. Various conditions for the Yamaguchi macrolactonization are applied to close the individual macrocycles. Finally a carefully elaborated endgame was developed to solve the problem of acyl migration in the case of neolaulimalide. All compounds were tested against several cell lines. The cytotoxicity of neolaulimalide could be confirmed for the first time since its original isolation and it could be shown that it induces tubulin polymerization as efficiently as laulimalide.

Primary cutaneous CD4+ small-/medium-sized pleomorphic T-cell lymphoma: a cutaneous nodular proliferation of pleomorphic T lymphocytes of undetermined significance? A study of 136 cases

Patients with skin nodules characterized by the infiltrate of pleomorphic small/medium T lymphocytes are currently classified as "primary cutaneous CD4+ small-/medium-sized pleomorphic T-cell lymphoma" (SMPTCL) or as T-cell pseudolymphoma. The distinction is often arbitrary, and patients with similar clinicopathologic features have been included in both groups. We studied 136 patients (male:female = 1:1; median age: 53 years, age range: 3-90 years) with cutaneous lesions that could be classified as small-/medium-sized pleomorphic T-cell lymphoma according to current diagnostic criteria. All but 3 patients presented with solitary nodules located mostly on the head and neck area (75%). Histopathologic features were characterized by nonepidermotropic, nodular, or diffuse infiltrates of small- to medium-sized pleomorphic T lymphocytes. A monoclonal rearrangement of the T-cell receptor-gamma gene was found in 60% of tested cases. Follow-up data available for 45 patients revealed that 41 of them were alive without lymphoma after a median time of 63 months (range: 1-357 months), whereas 4 were alive with cutaneous disease (range: 2-16 months). The incongruity between the indolent clinical course and the worrying histopathologic and molecular features poses difficulties in classifying these cases unambiguously as benign or malignant, and it may be better to refer to them with a descriptive term such as "cutaneous nodular proliferation of pleomorphic T lymphocytes of undetermined significance," rather than forcing them into one or the other category. On the other hand, irrespective of the name given to these equivocal cutaneous lymphoid proliferations, published data support a nonaggressive therapeutic strategy, particularly for patients presenting with solitary lesions.

Neural Networks as Cybernetic Systems - 3rd and revised edition

This third edition essentially compares with the 2nd one, but has been improved by correction of errors and by a rearrangement and minor expansion of the sections referring to recurrent networks. These changes hopefully allow for an easier comprehension of the essential aspects of this important domain that has received growing attention during the last years.

Neural Networks as Cybernetic Systems (3rd and revised edition) - Part I

Neural Networks as Cybernetic Systems is a textbox that combines classical systems theory with artificial neural network technology. This third edition essentially compares with the 2nd one, but has been improved by correction of errors and by a rearrangement and minor expansion of the sections referring to recurrent networks. These changes hopefully allow for an easier comprehension of the essential aspects of this important domain that has received growing attention during the last years.

Neural Networks as Cybernetic Systems (3rd and revised edition) - Part 2

eural Networks as Cybernetic Systems is a textbox that combines classical systems theory with artificial neural network technology. This third edition essentially compares with the 2nd one, but has been improved by correction of errors and by a rearrangement and minor expansion of the sections referring to recurrent networks. These changes hopefully allow for an easier comprehension of the essential aspects of this important domain that has received growing attention during the last years.

Intussusceptive angiogenesis: its role in embryonic vascular network formation

Intussusceptive angiogenesis is a novel mode of blood vessel formation and remodeling, which occurs by internal division of the preexisting capillary plexus without sprouting. In this study, the process is demonstrated in developing chicken eye vasculature and in the chorioallantoic membrane by methylmethacrylate (Mercox) casting, transmission electron microscopy, and in vivo observation. In a first step of intussusceptive angiogenesis, the capillary plexus expands by insertion of numerous transcapillary tissue pillars, ie, by intussusceptive microvascular growth. In a subsequent step, a vascular tree arises from the primitive capillary plexus as a result of intussusceptive pillar formation and pillar fusions, a process we termed "intussusceptive arborization." On the basis of the morphological observations, a 4-step model for intussusceptive arborization is proposed, as follows: phase I, numerous circular pillars are formed in rows, thus demarcating future vessels; phase II, formation of narrow tissue septa by pillar reshaping and pillar fusions; phase III, delineation, segregation, growth, and extraction of the new vascular entity by merging of septa; and phase IV, formation of new branching generations by successively repeating the process, complemented by growth and maturation of all components. In contrast to sprouting, intussusceptive angiogenesis does not require intense local endothelial cell proliferation; it is implemented primarily by rearrangement and attenuation of the endothelial cell plates. In summary, transcapillary pillar formation, ie, intussusception, is a central and probably widespread process, which plays a role not only in capillary network growth and expansion (intussusceptive microvascular growth), but also in vascular plexus remodeling and tree formation (intussusceptive arborization).

In situ dehydration behavior of veszelyite (Cu,Zn)2Zn(PO4)(OH)3·2H2O: A single-crystal X-ray study

The rare mixed copper-zinc phosphate mineral veszelyite (Cu,Zn)2Zn(PO4)(OH)3·2H2O space group P21/c, a = 7.5096(2), b = 10.2281(2), c = 9.8258(2) Å, β = 103.3040(10)°, V = 734.45(3) Å3 was investigated by in situ temperature-dependent single-crystal X-ray structure refinements. The atomic arrangement of veszelyite consists of an alternation of octahedral and tetrahedral sheets. The Jahn-Teller distorted CuO6 octahedra form sheets with eight-membered rings. The tetrahedral sheet composed of PO4 and ZnO3(OH) tetrahedra shows strong topological similarities to that of cavansite, gismondine, and kipushite.Diffraction data of a sample from Zdravo Vrelo, near Kreševo (Bosnia and Herzegovina) have been measured in steps of 25 up to 225 °C. Hydrogen positions and the hydrogen-bond system were determined experimentally from the structure refinements of data collected up to 125 °C. At 200 °C, the hydrogen-bonding scheme was inferred from bond-valence calculations and donor-acceptor distances. The hydrogen-bond system connects the tetrahedral sheet to the octahedral sheet and also braces the Cu sheet.At 150 °C, the H2O molecule at H2O2 was released and the Cu coordination (Cu1 and Cu2) decreased from originally six- to fivefold. Cu1 has a square planar coordination by four OH groups and an elongate distance to O3, whereas Cu2 has the Jahn-Teller characteristic elongate bond to H2O1. The unit-cell volume decreased 7% from originally 734.45(3) to 686.4(4) Å3 leading to a formula with 1 H2O pfu. The new phase observed above 150 °C is characterized by an increase of the c axis and a shortening of the b axis. The bending of T-O-T angles causes an increasing elliptical shape of the eight-membered rings in the tetrahedral and octahedral sheets. Moreover a rearrangement of the hydrogen-bond system was observed.At 225 °C, the structure degrades to an X-ray amorphous residual due to release of the last H2O molecule at H2O1. The stronger Jahn-Teller distortion of Cu1 relative to Cu2 suggests that Cu1 is fully occupied by Cu, whereas Cu2 bears significant Zn. H2O1 is the fifth ligand of Cu2. Zn at Cu2 is not favorable to adopt planar fourfold coordination. Thus, if the last water molecule is expelled the structure is destabilized.This study contributes to understanding the dehydration mechanism and thermal stability of supergene minerals characterized by Jahn-Teller distorted octahedra with mixed Cu, Zn occupancy.

Diagnostic and prognostic implications of the PAX8-PPARγ translocation in thyroid carcinomas-a TMA-based study of 226 cases

AIMS Follicular thyroid carcinoma (FTC) has been a diagnostic challenge for decades. The PAX8-PPARγ rearrangement has been detected in FTC and classic papillary thyroid carcinomas (PTCs). The aims of this study were to assess the presence of PAX8-PPARγ by using tissue microarrays in a large cohort of different thyroid neoplasms, and to assess its diagnostic and prognostic implications. METHODS AND RESULTS Fluorescence in-situ hybridization (FISH) analysis for PAX8-PPARγ was performed on 226 thyroid tumours, comprising FTCs (n = 59), PTCs (n = 126), poorly differentiated thyroid carcinomas (PDs; n = 34), follicular thyroid adenomas (FTAs; n = 5), and follicular tumours of unknown malignant potential (FTUMPs; n = 2). PAX8-PPARγ was detected in 12% of FTCs, 1% of PTCs, 7% of PDs, and in both cases of FTUMP. There was no correlation between the extent of capsular or vascular invasion and PAX8-PPARγ, or between lymph node or haematogenous metastasis and PAX8-PPARγ. Overall survival (OS), tumour-specific survival (TSS) and relapse-free-survival (RFS) were not influenced by PAX8-PPARγ. CONCLUSIONS In this study, we demonstrate for the first time the presence of PAX8-PPARγ in PDs and FTUMPs, whereas in FTCs and PTCs the prevalence of PAX8-PPARγ is lower than previously reported. PAX8-PPARγ did not correlate with invasiveness or affect prognosis in any tumour type.

Laser-induced transient grating analysis of dynamics of interaction between sensory rhodopsin II D75N and the HtrII transducer.

The interaction between sensory rhodopsin II (SRII) and its transducer HtrII was studied by the time-resolved laser-induced transient grating method using the D75N mutant of SRII, which exhibits minimal visible light absorption changes during its photocycle, but mediates normal phototaxis responses. Flash-induced transient absorption spectra of transducer-free D75N and D75N joined to 120 amino-acid residues of the N-terminal part of the SRII transducer protein HtrII (DeltaHtrII) showed only one spectrally distinct K-like intermediate in their photocycles, but the transient grating method resolved four intermediates (K(1)-K(4)) distinct in their volumes. D75N bound to HtrII exhibited one additional slower kinetic species, which persists after complete recovery of the initial state as assessed by absorption changes in the UV-visible region. The kinetics indicate a conformationally changed form of the transducer portion (designated Tr*), which persists after the photoreceptor returns to the unphotolyzed state. The largest conformational change in the DeltaHtrII portion was found to cause a DeltaHtrII-dependent increase in volume rising in 8 micros in the K(4) state and a drastic decrease in the diffusion coefficient (D) of K(4) relatively to those of the unphotolyzed state and Tr*. The magnitude of the decrease in D indicates a large structural change, presumably in the solvent-exposed HAMP domain of DeltaHtrII, where rearrangement of interacting molecules in the solvent would substantially change friction between the protein and the solvent.

The Oxytricha trifallax macronuclear genome: a complex eukaryotic genome with 16,000 tiny chromosomes.

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor "silent" germline micronuclear genome by a process of "unscrambling" and fragmentation. The tiny macronuclear "nanochromosomes" typically encode single, protein-coding genes (a small portion, 10%, encode 2-8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease.

Selective elevation of c-{\it myc} RNA transcripts during clonal evolution of N-methyl-N-nitrosourea induced thymic lymphomas in AKR/J mice

Untreated AKR mice develop spontaneous thymic lymphomas by 6-12 months of age. Lymphoma development is accelerated when young mice are injected with the carcinogen N-methyl-N-nitrosourea (MNU). Selected molecular and cellular events were compared during the latent period preceding "spontaneous" (retrovirally-induced) and MNU-induced thymic lymphoma development in AKR mice. These studies were undertaken to test the hypothesis that thymic lymphomas induced in the same inbred mouse strain by endogenous retroviruses and by a chemical carcinogen develop by different mechanisms.^ Immunofluorescence analysis of differentiation antigens showed that most MNU-induced lymphomas express an immature CD4-8+ profile. In contrast, spontaneous lymphomas represent each of the major lymphocyte subsets. These data suggest involvement of different target populations in MNU-induced and spontaneous lymphomas. Analyses at intervals after MNU treatment revealed selective expansion of the CD4-8+ J11d+ thymocyte subset at 8-10 weeks post-MNU in 68% of the animals examined, suggesting that these cells are targets for MNU-induced lymphomagenesis. Untreated age-matched animals showed no selective expansion of thymocyte subsets.^ Previous data have shown that both spontaneous and MNU-induced lymphomas are monoclonal or oligoclonal. Distinct rearrangement patterns of the J$\sb2$ region of the T-cell receptor $\beta$-chain showed emergence of clonal thymocyte populations beginning at 6-7 weeks after MNU treatment. However, lymphocytes from untreated animals showed no evidence of clonal expansion at the time intervals investigated.^ Activation of c-myc frequently occurs during development of B- and T- cell lymphomas. Both spontaneous and MNU-induced lymphomas showed increased c-myc transcript levels. Increased c-myc transcription was first detected at 6 weeks post-MNU, and persisted throughout the latent period. However, untreated animals showed no increases in c-myc transcripts at the time intervals examined. Another nuclear oncogene, c-fos, did not display a similar change in RNA transcription during the latent period.^ These results supports the hypothesis that MNU-induced and spontaneous tumors develop by multi-step pathways which are distinct with respect to the target cell population affected. Clonal emergence and c-myc deregulation are important steps in the development of both MNU-induced and spontaneous tumors, but the onset of these events is later in spontaneous tumor development. ^

Control of chromosomal rearrangements in {\it Escherichia coli\/}

A complete physical map of Escherichia coli K-12 strain MG1655 was constructed by digesting chromosomal DNA with the infrequently cutting restriction enzymes NotI, SfiI and XbaI and separating the fragments by pulsed field gel electrophoresis. The map was used to compare six K-12 strains of E. coli. Although several differences were noted and localized, the map of MG1655 was representative of all the K-12 strains tested. The maps were also used to analyze chromosomal rearrangements in the E. coli strain MG1655. The spontaneous and UV induced frequencies of tandem duplication formation were measured at several loci distributed around the chromosome. The spontaneous duplication frequency varied from 10$\sp{-5}$ to 10$\sp{-3}$ and increased at least ten-fold following mild UV irradiation treatment. Duplications of several regions of the chromosome, including the serA region and the metE region, were mapped using pulsed field gel electrophoresis. Duplications of serA were found to be large, ranging in size from 600 kb to 2100 kb. Several of the duplications isolated at serA were caused by ectopic recombination between IS5 elements and between IS186 elements. Duplications of the metE region, however, were almost exclusively the result of ectopic recombination between ribosomal RNA cistrons. Duplication frequencies were determined at both serA and metE in wild type and mismatch repair mutant strains (mutL, mutS, uvrD and recF). Even though all of the mismatch repair mutations increased duplication frequency of metE, the largest increases were observed in the mutL and mutS strains. Duplication frequency of serA was increased less dramatically by mutations in mismatch repair. Several duplications of metE isolated in a wild type and a mismatch repair mutant were mapped. The results showed that the same repeated sequences were used for duplication formation in the mismatch repair mutant as were used in the wild type strain. Several isolates showed evidence of multiple rearrangements indicating that mismatch repair may play a role in stabilizing the genome by controlling chromosomal rearrangement. ^