Analysis of Mice with Single and Multiple Copies of Transgenes Reveals a Novel Arrangement for the λ5-VpreB1 Locus Control Region

The murine λ5-V_preB1 locus encodes two proteins that form part of the pre-B-cell receptor and play a key role in B-lymphocyte development. We have identified a locus control region (LCR) which is responsible for coordinate activation of both genes in pre-B cells. Analysis of mice with single and multiple copies of transgenes shows a clear difference in the expression behavior of the genes depending on the transgene copy number. While expression of both λ5 and V_preB1 in single- and two-copy integrations requires the presence of a set of DNase I hypersensitive sites located 3′ of the λ5 gene, small fragments containing the genes have LCR activity when arranged in multiple-copy tandem arrays, indicating that additional components of the LCR are located within or close to the genes. The complete LCR is capable of driving efficient copy-dependent expression of a λ5 gene in pre-B cells even when it is integrated into centomeric γ-satellite DNA. The finding that activation of expression of the locus by positively acting factors is fully dominant over the silencing effect of heterochromatin has implications for models for chromatin-mediated gene silencing during B-cell development.     

Life‐history responses of a freshwater rotifer to copper pollution

In organisms with dormant stages, life‐history responses to past pollution can be studied retrospectively. Here, we study such responses in a rotifer (Brachionus calyciflorus) from the once heavily copper‐polluted Lake Orta (Italy). We extracted resting eggs from sediments, established clonal lineages from hatchlings, and exposed newborns of these lineages to one of three copper concentrations that each mimicked a specific period in the lake''s pollution history. For each rotifer, we daily collected life‐table data. We then estimated treatment‐specific vital rates and used a stage‐structured population model to project population growth rate λ. We also estimated elasticities of λ to vital rates and contributions of vital rates to observed Δλ between copper treatments. As expected, λ decreased with increasing copper concentration. This decrease resulted mostly from a decline in juvenile survival rate (S_J ) and partly from a decline in the survival rate of asexually reproducing females (S_A ). Maturation rate, and with one exception fecundity, also declined but did not contribute consistently to Δλ. λ was most elastic to S_J and S_A , indicating that survival rates were under stronger selection than maturation rate and fecundity. Together, our results indicate that variation in juvenile survival is a key component in the rotifers’ copper response. The consistent decrease in S_J with increasing copper stress and the sensitivity of λ to that decrease also suggest that juvenile survival is a useful indicator of population performance under environmental pollution.     

The immunoglobulin κ locus of primates

The immunoglobulin κ genes of nonhuman primates were studied by using sequence information and hybridization probes derived from the human κ gene regions. The following results were obtained: (1) V_κ gene probes of the three major human κ subgroups hybridized to restriction nuclease digests of DNA from the chimpanzees Pan troglodytes (PTR) and Pan paniscus (PPA), the gorilla Gorilla gorilla (GGO), the orangutan Pongo pygmaeus (PPY), the macaque Macaca mulatta (MMU), the marmoset Callithrix geoffrei (CGE), and the bushbaby Galago demidovii (GDE), yielding patterns of decreasing similarity to the patterns of the human V_κ multigene family. (2) The C_κ gene segments of PTR, GGO, and PPY were 99.6, 97, and 93%, respectively, identical in sequence to the human C_κ gene. A V_κ gene in PTR, GGO, PPY, and MMU was 98, 96, 96, and 95%, respectively, identical to the most C_κ proximal V_κ gene, called B3. The other two J_κ---C_κ proximal V_κ genes in human, B1 and B2, hybridize to restriction fragments of sizes identical to that of DNA from humans and great apes. (3) The long-range restriction maps of the human (HSA), PTR, and GGO κ loci as established by pulsed-field gel electrophoresis (PFGE) are quite homologous. According to the maps, however, and to hybridization studies with 11 duplication-differentiating probes, there is only one copy of the locus in PTR and GGO. This means that the duplication of large parts of the κ locus as found in humans occurred after the branch-point of human and great ape evolution. Despite the high similarity in structural details, the κ locus of the chimpanzee probably comprises only about half as many V_κ genes as the human κ locus. (4) Comparative PFGE experiments indicate that a V_κ-orphon region on the long arm of chromosome 2 (cos108) was translocated by a pericentric inversion that occurred after the separation of the GGO and PPY from the HSA and PTR clades.     

The visual system of the alligator

The eye tissues and liver of the alligator contain vitamin A₁ alone. The retina contains rhodopsin, typical in absorption spectrum (λ_max 500 mµ); but synthesized in solution from neo-b retinene and opsin much more rapidly than are the frog, mammalian, or chicken rhodopsins previously examined. In this regard alligator rhodopsin resembles the rhodopsins and porphyropsins of fishes, all of which so far investigated are synthesized rapidly in solution. The rates of synthesis in vitro of frog and alligator rhodopsins are matched closely by the rates of rod dark adaptation in living frogs and alligators, measured electrophysiologically at the same temperature. Alligator rods dark-adapt, and alligator rhodopsin is synthesized in solution, at rates characteristically associated with cones and cone pigments in frogs, mammals, and birds.     

Simple Uniaxial and Uniform Biaxial Deformation of Nearly Isotropic Incompressible Tissues

A method is developed for analyzing in a unified manner both uniaxial and uniform biaxial strain data obtained from nearly isotropic tissues. The formulation is a direct application of nonlinear elasticity theory pertaining to large deformations. The general relation between Eulerian stress (σ) and extension ratio (λ) in soft isotropic elastic bodies undergoing uniform deformation takes the simple form: σ = ((λ³ - 1)/λ) f(λ), where f(λ) must be determined for each material. The extension ratio may be either greater than 1.0 (uniaxial elongation), or lie between zero and 1.0 (uniform biaxial extension). Simple analytical functions for f(λ) are most readily found for each tissue by plotting all data as (λ³ - 1)/λσ vs. λ. Of those tissues investigated in this way (dog pericardium and pleura, and cat mesentery and dura), all but pleura could be adequately described by a parabola: 1/f(λ) = 1/k{[(λ_M - λ)(λ - λ_m)]/[λ_M - λ_m}. In these instances, three material constants per tissue (K, λ_M, λ_m) served to predict approximately the stresses attained during both small and large deformations, in strips and sheets alike. It was further found that the uniaxial strain asymptote (λ_M) was linearly related to the biaxial strain asymptote (Λ_M), thus effectively reducing the number of constants by one.     

Characterization of interactions within the Igα/Igβ transmembrane domains of the human B-cell receptor provides insights into receptor assembly

The B-cell receptor (BCR), a complex comprised of a membrane-associated immunoglobulin and the Igα/β heterodimer, is one of the most important immune receptors in humans and controls B-cell development, activity, selection, and death. BCR signaling plays key roles in autoimmune diseases and lymphoproliferative disorders, yet, despite the clinical significance of this protein complex, key regions (i.e., the transmembrane domains) have yet to be structurally characterized. The mechanism for BCR signaling also remains unclear and has been variously described by the mutually exclusive cross-linking and dissociation activation models. Common to these models is the significance of local plasma membrane composition, which implies that interactions between BCR transmembrane domains (TMDs) play a role in receptor functionality. Here we used an in vivo assay of TMD oligomerization called GALLEX alongside spectroscopic and computational methods to characterize the structures and interactions of human Igα and Igβ TMDs in detergent micelles and natural membranes. We observed weak self-association of the Igβ TMD and strong self-association of the Igα TMD, which scanning mutagenesis revealed was entirely stabilized by an E–X₁₀–P motif. We also demonstrated strong heterotypic interactions between the Igα and Igβ TMDs both in vitro and in vivo, which scanning mutagenesis and computational models suggest is multiconfigurational but can accommodate distinct interaction sites for self-interactions and heterotypic interactions of the Igα TMD. Taken together, these results demonstrate that the TMDs of the human BCR are sites of strong protein–protein interactions that may direct BCR assembly, endoplasmic reticulum retention, and immune signaling.     

A Residue-specific Shift in Stability and Amyloidogenicity of Antibody Variable Domains

Variable (V) domains of antibodies are essential for antigen recognition by our adaptive immune system. However, some variants of the light chain V domains (V_L) form pathogenic amyloid fibrils in patients. It is so far unclear which residues play a key role in governing these processes. Here, we show that the conserved residue 2 of V_L domains is crucial for controlling its thermodynamic stability and fibril formation. Hydrophobic side chains at position 2 stabilize the domain, whereas charged residues destabilize and lead to amyloid fibril formation. NMR experiments identified several segments within the core of the V_L domain to be affected by changes in residue 2. Furthermore, molecular dynamic simulations showed that hydrophobic side chains at position 2 remain buried in a hydrophobic pocket, and charged side chains show a high flexibility. This results in a predicted difference in the dissociation free energy of ∼10 kJ mol⁻¹, which is in excellent agreement with our experimental values. Interestingly, this switch point is found only in V_L domains of the κ family and not in V_Lλ or in V_H domains, despite a highly similar domain architecture. Our results reveal novel insight into the architecture of variable domains and the prerequisites for formation of amyloid fibrils. This might also contribute to the rational design of stable variable antibody domains.     

Correlation of Immunoglobulin G Expression and Histological Subtype and Stage in Breast Cancer

Introduction

Recently, growing evidence indicates that immunoglobulins (Igs) are not only produced by mature B lymphocytes or plasma cells, but also by various normal cells types at immune privileged sites and neoplasm, including breast cancer. However, the association of breast cancer derived IgG with genesis and development of the disease has not yet been established.

Methods

In this study we examined the expression of IgG in 186 breast cancers, 20 benign breast lesions and 30 normal breast tissues. Both immunohistochemistry with antibodies to Igκ (immunoglobulin G κ light chain) and Igγ (immunoglobulin G heavy chain) and in situ hybridization with an antisense probe to IgG1 heavy chain constant region gene were performed. Various clinicopathological features were also analyzed.

Results

We found that IgG is specifically expressed in human breast cancer cells. Both infiltrating ductal carcinoma and infiltrating lobular carcinoma had significantly greater numbers of Igκ and Igγ positive cancer cells as compared with medullary carcinoma, carcinoma in situ, and benign lesions (all p<0.05). In addition, IgG expression was correlated with breast cancer histological subtypes (p<0.01) and AJCC stages (p<0.05), with more abundance of IgG expression in more malignant histological subtypes or in more advanced stage of the disease.

Conclusions

IgG expression in breast cancer cells is correlated with malignancy and AJCC stages of the cancers. This suggests that breast cancer derived IgG may be associated with genesis, development and prognosis of the cancer.     

Thermal Characterization,Crystal Field Analysis and In-Band Pumped Laser Performance of Er Doped NaY(WO4)2 Disordered Laser Crystals

Undoped and Er-doped NaY(WO₄)₂ disordered single crystals have been grown by the Czochralski technique. The specific heat and thermal conductivity (κ) of these crystals have been characterized from T = 4 K to 700 K and 360 K, respectively. It is shown that κ exhibits anisotropy characteristic of single crystals as well as a κ(T) behavior observed in glasses, with a saturation mean free phonon path of 3.6 Å and 4.5 Å for propagation along a and c crystal axes, respectively. The relative energy positions and irreducible representations of Stark Er³⁺ levels up to ⁴G_7/2 multiplet have been determined by the combination of experimental low (<10 K) temperature optical absorption and photoluminescence measurements and simulations with a single-electron Hamiltonian including both free-ion and crystal field interactions. Absorption, emission and gain cross sections of the ⁴I_13/2↔⁴I_15/2 laser related transition have been determined at 77 K. The ⁴I_13/2 Er³⁺ lifetime (τ) was measured in the temperature range of 77–300 K, and was found to change from τ (77K) ≈ 4.5 ms to τ (300K) ≈ 3.5 ms. Laser operation is demonstrated at 77 K and 300 K by resonantly pumping the ⁴I_13/2 multiplet at λ≈1500 nm with a broadband (FWHM≈20 nm) diode laser source perfectly matching the 77 K crystal ⁴I_15/2 → ⁴I_13/2 absorption profile. At 77 K as much as 5.5 W of output power were obtained in π-polarized configuration with a slope efficiency versus absorbed pump power of 57%, the free running laser wavelength in air was λ≈1611 nm with the laser output bandwidth of 3.5 nm. The laser emission was tunable over 30.7 nm, from 1590.7 nm to 1621.4 nm, for the same π-polarized configuration.     

Entamoeba histolytica Cysteine Proteinase 5 Binds Integrin on Colonic Cells and Stimulates NFκB-mediated Pro-inflammatory Responses

Integrins are important mammalian receptors involved in normal cellular functions and the pathogenesis of inflammation and disease. Entamoeba histolytica is a protozoan parasite that colonizes the gut, and in 10% of infected individuals, causes amebic colitis and liver abscess resulting in 10⁵ deaths/year. E. histolytica-induced host inflammatory responses are critical in the pathogenesis of the disease, yet the host and parasite factors involved in disease are poorly defined. Here we show that pro-mature cysteine proteinase 5 (PCP5), a major virulent factor that is abundantly secreted and/or present on the surface of ameba, binds via its RGD motif to α_Vβ₃ integrin on Caco-2 colonic cells and stimulates NFκB-mediated pro-inflammatory responses. PCP5 RGD binding to α_Vβ₃ integrin triggered integrin-linked kinase(ILK)-mediated phosphorylation of Akt-473 that bound and induced the ubiquitination of NF-κB essential modulator (NEMO). As NEMO is required for activation of the IKKα-IKKβ complex and NFκB signaling, these events markedly up-regulated pro-inflammatory mediator expressions in vitro in Caco-2 cells and in vivo in colonic loop studies in wild-type and Muc2^−/− mice lacking an intact protective mucus barrier. These results have revealed that EhPCP5 RGD motif is a ligand for α_Vβ₃ integrin-mediated adhesion on colonic cells and represents a novel mechanism that E. histolytica trophozoites use to trigger an inflammatory response in the pathogenesis of intestinal amebiasis.     

Cryptochrome, Phytochrome, and the Photoregulation of Anthocyanin Production under Blue Light

The principle of equivalent light action predicts that two light treatments (wavelengths ^λ₁ and λ₂) producing the same Pfr/P ratio (_λ1 = _λ2) and the same rate of phytochrome photoconversion (k_λ1 = k_λ2) are perceived by phytochrome as being the same and should produce the same effect. The results of experiments based on the principle of equivalent light action indicate that cryptochrome is involved in the photoregulation of anthocyanin production elicited by blue light in tomato seedlings. This was also the case for one strain of cabbage seedlings. For another strain of cabbage seedlings, the results suggest that cryptochrome is either not involved or that the state of phytochrome is the principal limiting factor.     

Genome analysis and signature discovery for diving and sensory properties of the endangered Chinese alligator

Crocodilians are diving reptiles that can hold their breath under water for long periods of time and are crepuscular animals with excellent sensory abilities. They comprise a sister lineage of birds and have no sex chromosome. Here we report the genome sequence of the endangered Chinese alligator (Alligator sinensis) and describe its unique features. The next-generation sequencing generated 314 Gb of raw sequence, yielding a genome size of 2.3 Gb. A total of 22 200 genes were predicted in Alligator sinensis using a de novo, homology- and RNA-based combined model. The genetic basis of long-diving behavior includes duplication of the bicarbonate-binding hemoglobin gene, co-functioning of routine phosphate-binding and special bicarbonate-binding oxygen transport, and positively selected energy metabolism, ammonium bicarbonate excretion and cardiac muscle contraction. Further, we elucidated the robust Alligator sinensis sensory system, including a significantly expanded olfactory receptor repertoire, rapidly evolving nerve-related cellular components and visual perception, and positive selection of the night vision-related opsin and sound detection-associated otopetrin. We also discovered a well-developed immune system with a considerable number of lineage-specific antigen-presentation genes for adaptive immunity as well as expansion of the tripartite motif-containing C-type lectin and butyrophilin genes for innate immunity and expression of antibacterial peptides. Multifluorescence in situ hybridization showed that alligator chromosome 3, which encodes DMRT1, exhibits significant synteny with chicken chromosome Z. Finally, population history analysis indicated population admixture 0.60-1.05 million years ago, when the Qinghai-Tibetan Plateau was uplifted.     

Secreted Heat Shock Protein 90α Induces Colorectal Cancer Cell Invasion through CD91/LRP-1 and NF-κB-mediated Integrin αV Expression

HCT-8 colon cancer cells secreted heat shock protein 90α (HSP90α) and had increased invasiveness upon serum starvation. The concentrated conditioned medium of serum-starved HCT-8 cells was able to stimulate the migration and invasion of non-serum-starved cells, which could be prevented by treatment with an anti-HSP90α antibody. Recombinant HSP90α (rHSP90α) also enhanced HCT-8 cell migration and invasion, suggesting a stimulatory role of secreted HSP90α in cancer malignancy. HSP90α binding to CD91α and Neu was evidenced by the proximity ligation assay, and rHSP90α-induced HCT-8 cell invasion could be suppressed by the addition of anti-CD91α or anti-Neu antibodies. Via CD91α and Neu, rHSP90α selectively induced integrin α_V expression, and knockdown of integrin α_V efficiently blocked rHSP90α-induced HCT-8 cell invasion. rHSP90α induced the activities of ERK, PI3K/Akt, and NF-κB p65, but only NF-κB activation was involved in HSP90α-induced integrin α_V expression. Additionally, we investigated the serum levels of HSP90α and the expression status of tumor integrin α_V mRNA in colorectal cancer patients. Serum HSP90α levels of colorectal cancer patients were significantly higher than those of normal volunteers (p < 0.001). Patients with higher serum HSP90α levels significantly exhibited elevated levels of integrin α_V mRNA in tumor tissues as compared with adjacent non-tumor tissues (p < 0.001). Furthermore, tumor integrin α_V overexpression was significantly correlated with TNM (Tumor, Node, Metastasis) staging (p = 0.001).     

Sulfohydrolase Activity and Carrageenan Biosynthesis in Chondrus crispus (Rhodophyceae)

An enzyme catalyzing the conversion of μ- to κ-carrageenan has been demonstrated in both haploid and diploid plants of Chondrus crispus. It acts at the polymer level producing 3,6-anhydro-d-galactose with the stoichiometric release of sulfate. Two-thirds of the recoverable enzyme was associated with the 15,000g pellet most of which could be solubilized by passage through a Ribi Cell Fractionator. The enzyme precipitated between 2.65 and 4.24 m (NH₄)₂SO₄ and was partly purified on DEAE-cellulose columns. This sulfohydrolase has a pH optimum near 6.5 and is inhibited by molybdate, phosphate, sulfate, tungstate, cysteine, ATP, GTP, UDP, and by λ-carrageenan. No activator was found. The enzyme showed a similar affinity for several preparations of μ-carrageenan and for the κ-carrageenase-resistant fraction from κ-carrageenan thus confirming that the latter is a biosynthetically unfinished molecule.