Mutation in beta1-tubulin correlates with macrothrombocytopenia in Cavalier King Charles Spaniels.

BACKGROUND Cavalier King Charles Spaniels (CKCS) have a high prevalence of inherited macrothrombocytopenia. The purpose of this study was to determine if a mutation in beta1-tubulin correlated with presumptive inherited macrothrombocytopenia. HYPOTHESIS A mutation in beta1-tubulin results in synthesis of an altered beta1-tubulin monomer. alpha-beta tubulin dimers within microtubule protofilaments are unstable, resulting in altered megakaryocyte proplatelet formation. ANIMALS Blood samples were obtained from CKCS and non-CKCS dogs. METHODS DNA was used in polymerase chain reaction (PCR) assays to evaluate beta1-tubulin. Platelet numbers and mean platelet volume (MPV) were evaluated for a correlation with the presence or absence of a mutation identified in beta1-tubulin. Platelets obtained from homozygous, heterozygous, and clear CKCS were further evaluated using electron microscopy and immunofluorescence. RESULTS A mutation in the gene encoding beta1-tubulin correlated with macrothrombocytopenia in CKCS. Electron microscopy and immunofluorescence studies suggest that platelet microtubules are present but most likely are unstable and decreased in number. CONCLUSIONS AND CLINICAL IMPORTANCE The macrothrombocytopenia of CKCS correlated with a mutation in beta1-tubulin. alpha-beta tubulin dimers within protofilaments most likely are unstable, leading to altered proplatelet formation by megakaryocytes. This information will aid in distinguishing inherited from acquired thrombocytopenia. It also provides insight into the mechanism of platelet production by megakaryocytes, and also may prove useful in understanding heart-related changes in macrothrombocytopenic CKCS with concurrent mitral valve regurgitation.

Liquid–Vapor Interface of Formic Acid Solutions in Salt Water: A Comparison of Macroscopic Surface Tension and Microscopic in Situ X-ray Photoelectron Spectroscopy Measurements

The liquid–vapor interface is difficult to access experimentally but is of interest from a theoretical and applied point of view and has particular importance in atmospheric aerosol chemistry. Here we examine the liquid–vapor interface for mixtures of water, sodium chloride, and formic acid, an abundant chemical in the atmosphere. We compare the results of surface tension and X-ray photoelectron spectroscopy (XPS) measurements over a wide range of formic acid concentrations. Surface tension measurements provide a macroscopic characterization of solutions ranging from 0 to 3 M sodium chloride and from 0 to over 0.5 mole fraction formic acid. Sodium chloride was found to be a weak salting out agent for formic acid with surface excess depending only slightly on salt concentration. In situ XPS provides a complementary molecular level description about the liquid–vapor interface. XPS measurements over an experimental probe depth of 51 Å gave the C 1s to O 1s ratio for both total oxygen and oxygen from water. XPS also provides detailed electronic structure information that is inaccessible by surface tension. Density functional theory calculations were performed to understand the observed shift in C 1s binding energies to lower values with increasing formic acid concentration. Part of the experimental −0.2 eV shift can be assigned to the solution composition changing from predominantly monomers of formic acid to a combination of monomers and dimers; however, the lack of an appropriate reference to calibrate the absolute BE scale at high formic acid mole fraction complicates the interpretation. Our data are consistent with surface tension measurements yielding a significantly more surface sensitive measurement than XPS due to the relatively weak propensity of formic acid for the interface. A simple model allowed us to replicate the XPS results under the assumption that the surface excess was contained in the top four angstroms of solution.

Excitonic Splitting, Delocalization, and Vibronic Quenching in the Benzonitrile Dimer

The excitonic S1/S2 state splitting and the localization/delocalization of the S1 and S2 electronic states are investigated in the benzonitrile dimer (BN)2 and its 13C and d5 isotopomers by mass-resolved two-color resonant two-photon ionization spectroscopy in a supersonic jet, complemented by calculations. The doubly hydrogen-bonded (BN-h5)2 and (BN-d5)2 dimers are C2h symmetric with equivalent BN moieties. Only the S0 → S2 electronic origin is observed, while the S0 → S1 excitonic component is electric-dipole forbidden. A single 12C/13C or 5-fold h5/d5 isotopic substitution reduce the dimer symmetry to Cs, so that the heteroisotopic dimers (BN)2-(h5 – h513C), (BN)2-(h5 – d5), and (BN)2-(h5 – h513C) exhibit both S0 → S1 and S0 → S2 origins. Isotope-dependent contributions Δiso to the excitonic splittings arise from the changes of the BN monomer zero-point vibrational energies; these range from Δiso(12C/13C) = 3.3 cm–1 to Δiso(h5/d5) = 155.6 cm–1. The analysis of the experimental S1/S2 splittings of six different isotopomeric dimers yields the S1/S2 exciton splitting Δexc = 2.1 ± 0.1 cm–1. Since Δiso(h5/d5) ≫ Δexc and Δiso(12C/13C) > Δexc, complete and near-complete exciton localization occurs upon 12C/13C and h5/d5 substitutions, respectively, as diagnosed by the relative S0 → S1 and S0 → S2 origin band intensities. The S1/S2 electronic energy gap of (BN)2 calculated by the spin-component scaled approximate second-order coupled-cluster (SCS-CC2) method is Δelcalc = 10 cm–1. This electronic splitting is reduced by the vibronic quenching factor Γ. The vibronically quenched exciton splitting Δelcalc·Γ = Δvibroncalc = 2.13 cm–1 is in excellent agreement with the observed splitting Δexc = 2.1 cm–1. The excitonic splittings can be converted to semiclassical exciton hopping times; the shortest hopping time is 8 ps for the homodimer (BN-h5)2, the longest is 600 ps for the (BN)2(h5 – d5) heterodimer.

Syntheses, structures and magnetic properties of two copper(Ii) diphosphonates: [NH3(CH2)2NH3]2[Cu2(hedp)2]·H2O and [NH3CH(CH3)CH2NH3]2[Cu2(hedp)2] (hedp = 1-hydroxyethylidenediphosphonate)

This paper describes the syntheses and characterization of two new copper(II) diphosphonates: [NH3(CH2)2NH3]2[Cu2(hedp)2]·H2O (1) and [NH3CH(CH3)CH2NH3]2[Cu2(hedp)2] (2) (hedp = 1-hydroxyethylidenediphosphonate). Both compounds exhibit similar one-dimensional linear chain structures. The symmetrical {Cu2(hedp)2} dimers are connected by edge-shared {CuO5} square pyramids and form infinite chains. The Cu(II) ions are alternately bridged by O–P–O groups and O atoms. The Cu–O–Cu angles are 95.8(1) and 96.1(1)° for 1 and 2, respectively. Their magnetic properties show moderately strong antiferromagnetic interactions in both compounds.

Transgenic Expression of Human CD46 on Porcine Endothelium: Effect on Coagulation and Fibrinolytic Cascades During Ex Vivo Human-to-Pig Limb Xenoperfusions

BACKGROUND Dysregulation of the coagulation system due to inflammatory responses and cross-species molecular incompatibilities represents a major obstacle to successful xenotransplantation. We hypothesized that complement inhibition mediated by transgenic expression of human CD46 in pigs might also regulate the coagulation and fibrinolysis cascades and tested this in ex vivo human-to-pig xenoperfusions. METHODS Forelimbs of wild-type and hCD46/HLA-E double transgenic pigs were ex vivo xenoperfused for 12 hours with whole heparinized human blood. Muscle biopsies were stained for galactose-α1,3-galactose, immunoglobulin M, immunoglobulin G, complement, fibrin, tissue factor, fibrinogen-like protein 2, tissue plasminogen activator (tPA), and plasminogen activator inhibitor (PAI)-1. The PAI-1/tPA complexes, D-dimers, and prothrombin fragment F1 + 2 were measured in plasma samples after ex vivo xenoperfusion. RESULTS No differences of galactose expression or deposition of immunoglobulin M and immunoglobulin G were found in xenoperfused tissues of wild type and transgenic limbs. In contrast, significantly lower deposition of C5b-9 (P < 0.0001), fibrin (P = 0.009), and diminished expression of tissue factor (P = 0.005) and fibrinogen-like protein 2 (P = 0.028) were found in xenoperfused tissues of transgenic limbs. Levels of prothrombin fragment F1 + 2 (P = 0.031) and D-dimers (P = 0.044) were significantly lower in plasma samples obtained from transgenic as compared to wild-type pig limb perfusions. The expression of the fibrinolytic marker tPA was significantly higher (P = 0.009), whereas PAI-1 expression (P = 0.022) and PAI-1/tPA complexes in plasma (P = 0.015) were lower after transgenic xenoperfusion as compared to wild-type xenoperfusions. CONCLUSIONS In this human-to-pig xenoperfusion model, complement inhibition by transgenic hCD46 expression led to a significant inhibition of procoagulant and antifibrinolytic pathways.

Mass spectrometric study of molecular and ionic sublimation of lanthanum triiodide

The molecular and ionic composition of saturated vapor over lanthanum triiodide was studied by Knudsen effusion mass spectrometry. The (LaI3)n molecules (n = 1–3) and the [I(LaI3)n]− ions (n = 0–4) were observed. The partial pressures of the molecules were determined and the enthalpies of sublimation, ΔsH° (298.15 K) in kJ mol−1, in the form of monomers (304 ± 7), dimers (428 ± 25), and trimers (455 ± 50) were obtained by the second and third laws of thermodynamics. The enthalpy of formation, ΔfH° (298.15 K) in kJ mol−1, of the LaI3 (−376 ± 10), La2I6 (−932 ± 25), La3I9 (−1585 ± 50) molecules and the LaI4− (−841 ± 24), La2I7− (−1486 ± 32) ions were determined. The electron work function, φe = 3.5 ± 0.3 eV, for the LaI3 crystal was calculated from the thermochemical cycle.

The elusive S2 state, the S1/S2 splitting, and the excimer states of the benzene dimer

We observe the weak S 0 → S 2 transitions of the T-shaped benzene dimers (Bz)2 and (Bz-d 6)2 about 250 cm−1 and 220 cm−1 above their respective S 0 → S 1 electronic origins using two-color resonant two-photon ionization spectroscopy. Spin-component scaled (SCS) second-order approximate coupled-cluster (CC2) calculations predict that for the tipped T-shaped geometry, the S 0 → S 2 electronic oscillator strength f el (S 2) is ∼10 times smaller than f el (S 1) and the S 2 state lies ∼240 cm−1 above S 1, in excellent agreement with experiment. The S 0 → S 1 (ππ ∗) transition is mainly localized on the “stem” benzene, with a minor stem → cap charge-transfer contribution; the S 0 → S 2 transition is mainly localized on the “cap” benzene. The orbitals, electronic oscillator strengths f el (S 1) and f el (S 2), and transition frequencies depend strongly on the tipping angle ω between the two Bz moieties. The SCS-CC2 calculated S 1 and S 2 excitation energies at different T-shaped, stacked-parallel and parallel-displaced stationary points of the (Bz)2 ground-state surface allow to construct approximate S 1 and S 2 potential energy surfaces and reveal their relation to the “excimer” states at the stacked-parallel geometry. The f el (S 1) and f el (S 2) transition dipole moments at the C 2v -symmetric T-shape, parallel-displaced and stacked-parallel geometries are either zero or ∼10 times smaller than at the tipped T-shaped geometry. This unusual property of the S 0 → S 1 and S 0 → S 2 transition-dipole moment surfaces of (Bz)2 restricts its observation by electronic spectroscopy to the tipped and tilted T-shaped geometries; the other ground-state geometries are impossible or extremely difficult to observe. The S 0 → S 1/S 2 spectra of (Bz)2 are compared to those of imidazole ⋅ (Bz)2, which has a rigid triangular structure with a tilted (Bz)2 subunit. The S 0 → S 1/ S 2 transitions of imidazole-(benzene)2 lie at similar energies as those of (Bz)2, confirming our assignment of the (Bz)2 S 0 → S 2 transition.

Structural elucidation and antisense properties of a sugar-modified DNA analogue

Antisense oligonucleotides deserve great attention as potential drug candidates for the treatment of genetic disorders. For example, muscle dystrophy can be treated successfully in mice by antisense-induced exon skipping in the pre-mRNA coding for the structural protein dystrophin in muscle cells. For this purpose a sugar- and backbone-modified DNA analogue was designed, in which a tricyclic ring system substitutes the deoxyribose. These chemical modifications stabilize the dimers formed with the targeted RNA relative to native nucleic acid duplexes and increase the biostability of the antisense oligonucleotide. While evading enzymatic degradation constitutes an essential property of antisense oligonucleotides for therapeutic application, it renders the oligonucleotide inaccessible to biochemical sequencing techniques and requires the development of alternative methods based on mass spectrometry. The set of sequences studied includes tcDNA oligonucleotides ranging from 10 to 15 nucleotides in length as well as their hybrid duplexes with DNA and RNA complements. All samples were analyzed on a LTQ Orbitrap XL instrument equipped with a nano-electrospray source. For tandem mass spectrometric experiments collision-induced dissociation was performed, using helium as collision gas. Mass spectrometric sequencing of tcDNA oligomers manifests the applicability of the technique to substrates beyond the scope of enzyme-based methods. Sequencing requires the formation of characteristic backbone fragments, which take the form of a-B- and w-ions in the product ion spectra of tcDNA. These types of product ions are typically associated with unmodified DNA, which suggests a DNA-like fragmentation mechanism in tcDNA. The loss of nucleobases constitutes the second prevalent dissociation pathway observed in tcDNA. Comparison of partially and fully modified oligonucleotides indicates a pronounced impact of the sugar-moiety on the base loss. As this event initiates cleavage of the backbone, the presented results provide new mechanistic insights into the fragmentation of DNA in the gas-phase. The influence of the sugar-moiety on the dissociation extends to tcDNA:DNA and tcDNA:RNA hybrid duplexes, where base loss was found to be much more prominent from sugar-modified oligonucleotides than from their natural complements. Further prominent dissociation channels are strand separation and backbone cleavage of the single strands, as well as the ejection of backbone fragments from the intact duplex. The latter pathway depends noticeably on the base sequence. Moreover, it gives evidence of the high stability of the hybrid dimers, and thus directly reflects the affinity of tcDNA for its target in the cell. As the cellular target of tcDNA is a pre-mRNA, the structure was designed to discriminate RNA from DNA complements, which could be demonstrated by mass spectrometric experiments.

High-resolution atomic force microscopy imaging of rhodopsin in rod outer segment disk membranes.

Atomic force microscopy (AFM) is a powerful imaging technique that allows recording topographical information of membrane proteins under near-physiological conditions. Remarkable results have been obtained on membrane proteins that were reconstituted into lipid bilayers. High-resolution AFM imaging of native disk membranes from vertebrate rod outer segments has unveiled the higher-order oligomeric state of the G protein-coupled receptor rhodopsin, which is highly expressed in disk membranes. Based on AFM imaging, it has been demonstrated that rhodopsin assembles in rows of dimers and paracrystals and that the rhodopsin dimer is the fundamental building block of higher-order structures.

Biomarker-guided personalised emergency medicine for all - hope for another hype?

Polymorbid patients, diverse diagnostic and therapeutic options, more complex hospital structures, financial incentives, benchmarking, as well as perceptional and societal changes put pressure on medical doctors, specifically if medical errors surface. This is particularly true for the emergency department setting, where patients face delayed or erroneous initial diagnostic or therapeutic measures and costly hospital stays due to sub-optimal triage. A "biomarker" is any laboratory tool with the potential better to detect and characterise diseases, to simplify complex clinical algorithms and to improve clinical problem solving in routine care. They must be embedded in clinical algorithms to complement and not replace basic medical skills. Unselected ordering of laboratory tests and shortcomings in test performance and interpretation contribute to diagnostic errors. Test results may be ambiguous with false positive or false negative results and generate unnecessary harm and costs. Laboratory tests should only be ordered, if results have clinical consequences. In studies, we must move beyond the observational reporting and meta-analysing of diagnostic accuracies for biomarkers. Instead, specific cut-off ranges should be proposed and intervention studies conducted to prove outcome relevant impacts on patient care. The focus of this review is to exemplify the appropriate use of selected laboratory tests in the emergency setting for which randomised-controlled intervention studies have proven clinical benefit. Herein, we focus on initial patient triage and allocation of treatment opportunities in patients with cardiorespiratory diseases in the emergency department. The following five biomarkers will be discussed: proadrenomedullin for prognostic triage assessment and site-of-care decisions, cardiac troponin for acute myocardial infarction, natriuretic peptides for acute heart failure, D-dimers for venous thromboembolism, C-reactive protein as a marker of inflammation, and procalcitonin for antibiotic stewardship in infections of the respiratory tract and sepsis. For these markers we provide an overview on physiopathology, historical evolution of evidence, strengths and limitations for a rational implementation into clinical algorithms. We critically discuss results from key intervention trials that led to their use in clinical routine and potential future indications. The rational for the use of all these biomarkers, first, tackle diagnostic ambiguity and consecutive defensive medicine, second, delayed and sub-optimal therapeutic decisions, and third, prognostic uncertainty with misguided triage and site-of-care decisions all contributing to the waste of our limited health care resources. A multifaceted approach for a more targeted management of medical patients from emergency admission to discharge including biomarkers, will translate into better resource use, shorter length of hospital stay, reduced overall costs, improved patients satisfaction and outcomes in terms of mortality and re-hospitalisation. Hopefully, the concepts outlined in this review will help the reader to improve their diagnostic skills and become more parsimonious laboratory test requesters.

The activation peptide of coagulation factor XIII is vital for its expression and stability.

BACKGROUND The human activation peptide of factor XIII (AP-FXIII) comprises the first 37 amino acids of the N-terminus and holds the FXIII in an inactive state. FXIII is activated either proteolytically by cleavage of AP-FXIII by thrombin, or non-proteolytically by high calcium concentrations. OBJECTIVE To investigate the role of AP-FXIII in the expression and stability of FXIII. METHODS We cloned 13 FXIII variants with progressive truncations of AP-FXIII from the N-terminus (delN-FXIII-A), expressed them in mammalian cells, and measured their thermostability, activation, and transglutaminase activity. We also used in silico calculations to analyze the stability of hypothetical delN-FXIII dimers and to identify crucial motifs within AP-FXIII. RESULTS Variants with deletions longer than the first 10 amino acids and an R11Q point mutant were not expressed as proteins. In silico calculations indicated that the sequence (8) FGGR(12) R plays a substantial role in intersubunit interactions in FXIII-A2 homodimers. In agreement with this prediction, the temperature stability of delN-FXIII variants decreased with increasing length of deletion. These results may suggest a role of the N-terminus of AP-FXIII in dimer stability. Substantial sequence homology was found among activation peptides of vertebrate and even invertebrate (crustacean) FXIII-A orthologs, which further supports our conclusion. CONCLUSIONS We conclude that deletion of 11 or more N-terminal amino acids disrupts intersubunit interactions, which may prevent FXIII-A2 homodimer formation. Therefore, AP-FXIII plays an important role in the stability of the FXIII-A2 dimer.

DNA damage-induced Fas ligand expression by epidermal dendritic cells mediates UV-induced immune suppression of contact hypersensitivity

Exposure to UVB radiation induces local and systemic immune suppression, evidenced by inhibition of the contact hypersensitivity response (CHS). Epidermal dendritic cells, the primary antigen presenting cells responsible for the induction of CHS, are profoundly altered in phenotype and function by UVB exposure and possess UV-specific DNA damage upon migrating to skin-draining lymph nodes. Expression of the proapoptotic protein FasL has been demonstrated in both skin and lymph node cells following UVB exposure. Additionally, functional FasL expression has recently been demonstrated to be required in the phenomenon of UV-induced immune suppression. To test the hypothesis that FasL expression by DNA-damaged Langerhans cells migrating to the skin-draining lymph nodes is a crucial event in the generation of this phenomenon, mice were given a single 5KJ/m2 UV-B exposure and sensitized to 0.5% FITC through the exposed area. Dendritic cells (DC) harvested from skin-draining lymph nodes (DLN) 18 hours following sensitization by magnetic CD11c-conjugated microbeads expressed high levels of Iab, CD80 and CD86, DEC-205 and bore the FITC hapten, suggesting epidermal origin. Radioimmunoassay of UV-specific DNA damage showed that DC contained the vast majority of cyclobutane pyrimidine dimers (CPDs) found in the DLN after UVB and exhibited increased FasL mRNA expression, a result which correlated with greatly increased FasL-mediated cytotoxicity. The ability of DCs to transfer sensitization to naïve hosts was lost following UVB exposure, a phenomenon which required DC FasL expression, and was completely reversed by cutaneous DNA repair. Collectively, these results demonstrate the central importance of DNA damage-induced FasL expression on migrating dendritic cells in mediating UV-induced suppression of contact hypersensitivity. ^

Homeostatic transcriptional regulation of bcl-2 in the epidermis

Bcl-2, a crucial regulator of cell survival, is frequently overexpressed in basal cell carcinomas (BCCs), the most commonly diagnosed cancers. Regulation of bcl-2 expression in epidermal keratinocytes is not well characterized. In the epidermis, bcl-2 is expressed only in keratinocytes of the basal layer and the outer root sheath of hair follicles and no bcl-2 expression in suprabasalar keratinocytes. The calcium gradient in the epidermis is a potent regulator of keratinocyte differentiation. Increasing calcium concentrations associated with differentiation, resulted in the downregulation of a 2.9 kb bcl-2 promoter luciferase construct. The AP-1 family of transcription factors is differentially expressed in the strata of the epidermis and has been shown to be involved in the stage specific expression of numerous differentiation markers in the epidermis. In silico analysis of the bcl-2 promoter and gene reporter assays showed that co-transfection of JUNB and JUND, but not other AP-1 dimers, caused a significant upregulation of the bcl-2 promoter in primary keratinocytes. Immunoelectrophoretic mobility shift assays, in vivo chromatin immunoprecipitation (ChIP) studies and mutational analysis of AP-1 binding site 3 on the bcl-2 promoter identified it as the site involved in bcl-2 regulation. Utilizing site directed mutants, we determined that phosphorylation at Ser90/Ser100 residues of JUND is required for the activation of the bcl-2 promoter. ^ The sonic hedgehog (SHH) pathway is frequently deregulated in BCCs and, we have shown that GLI1 upregulates bcl-2 in keratinocytes. While examining potential regulation of the SHH pathway extracellular calcium, we found that higher calcium concentrations are associated with lowered HH pathway activity and upregulation of suppressor of fused (SUFU) which negatively regulates the SHH pathway. ChIP assays, and in vivo mouse models, show that ΔNp63α, a crucial regulator of epidermal development, binds and activates the SUFU promoter in differentiating keratinocytes. Increasing SUFU levels prevent transactivation of the bcl-2 promoter. In vitro SUFU knockdown along with in vivo SUFU+/− murine models demonstrate a significant upregulation of bcl-2 expression. ^ In conclusion, the spatial and temporal expression of bcl-2 during keratinocyte differentiation in the epidermis is a complex process requiring cooperative interactions of specific signaling cascades and transcription factors. ^

Investigation of mechanisms involved in ultraviolet B radiation-induced, T cell -mediated immune suppression

Cutaneous exposure to ultraviolet-B radiation (UVR) results in the suppression of cell-mediated immune responses such as contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH). This modulation of immune responses is mediated by local or systemic mechanisms, both of which are associated with the generation of antigen-specific suppressor T lymphocytes (Ts). UV-induced Ts have been shown to be CD3+CD4+CD8 − T cells that control multiple immunological pathways. However, the precise mechanisms involved in the generation and function of these immunoregulatory cells remain unclear. We investigated the cellular basis for the generation of UV-induced Ts lymphocytes in both local and systemic models of immune suppression, and further examined the pleiotrophic function of these immunoregulatory cells. ^ We used Thy1.1 and Thy1.2 congenic mice in a draining lymph node (DLN) cell transfer model to analyze the role played by epidermal Langerhans cells in the generation of Ts cells. We demonstrate that T cells tightly adhered to antigen-presenting cells (APC) from UV-irradiated skin are the direct progenitors of UV-induced Ts lymphocytes. Our studies also reveal that UV-induced DNA-damage in the form of cyclobutyl pyrimidine dimers (CPD) in the epidermal APC is crucial for the altered maturation of these adherent T cells into Ts. ^ We used TCR transgenic mice in an adoptive transfer model and physically tracked the antigen-specific clones during immune responses in unirradiated versus UV-irradiated mice. We demonstrate that UV-induced Ts and effector TDTH cells share the same epitope specificity, indicating that both cell populations arise from the same clonal progenitors. UVR also causes profound changes in the localization and proliferation of antigen-specific T cells during an immune response. Antigen-specific T cells are not detectable in the DLNs of UV-irradiated mice after 3 days post-immunization, but are found in abundance in the spleen. In contrast, these clones continue to be found in the DLNs and spleens of normal animals several days post-immunization. Our studies also reveal that a Th2 cytokine environment is essential for the generation of Ts in UV-irradiated mice. ^ The third part of our study examined the pleiotrophic nature of UV-induced Ts. We used a model for the induction of both cellular and humoral responses to human gamma-globulin (HGG) to demonstrate that UV-induced Ts lymphocytes can suppress DTH as well as antibody responses. (Abstract shortened by UMI.) ^

L -Sox5 and Sox6: Architectural transcription factors in chondrogenesis

Transcription factors often determine cell fate and tissue development. Chondrogenesis is the developmental process by which cartilages form. Recently, gene targeting studies have shown that two transcription factors, L-Sox5 and Sox6, play essential and redundant roles in chondrogenesis in vivo by converting precartilaginous cell condensations into cartilages. Both are highly similar High-Mobility-Group (HMG)-domain proteins that bind and subsequently bend DNA containing the 7bp HMG site (A/T)(A/T)CAA(A/T)G. They have no transactivation domain, but homo- and hetero-dimerize and preferentially bind DNA containing two HMG sites. They are thought to play an architectural role in transactivation by facilitating long-range DNA and protein interactions. To understand their molecular mechanism of action, we investigated how phasing, orientation, and spacing between HMG sites affect L-Sox5 and Sox6 DNA-binding. We determined that L-Sox5 and Sox6 dimers bind with high affinity to paired HMG sites in DNA rather than a single HMG site. Binding of paired sites is independent of DNA helical phasing, orientation of paired HMG sites and independent of distance up to 255 base pairs between sites. Mutational analysis demonstrated that binding of L-Sox5 and Sox6, independent of orientation of the sites, is critically dependent on the presence of paired HMG sites rather than one HMG site alone. Our data support a unique and novel model whereby L-Sox5 and Sox6 dimerize and bind DNA with pronounced spatial flexibility, possibly by a flexible hinge, and act as architectural transcription factors that bring distant DNA sites and proteins together to form higher order transcriptional complexes that are essential for the activation of their target genes in chondrogenesis. ^