Analysis of DNA ligase IV mutations found in LIG4 syndrome patients: the impact of two linked polymorphisms

LIG4 syndrome patients have hypomorphic mutations in DNA ligase IV. Although four of the five identified patients display immunodeficiency and developmental delay, one patient was developmentally normal. The developmentally normal patient had the same homozygous mutation (R278H) in DNA ligase IV as one of the more severely affected patients, who additionally had two linked polymorphisms. Here, we examine the impact of the mutations and polymorphisms identified in the LIG4 syndrome patients. Examination of recombinant mutant proteins shows that the severity of the clinical features correlates with the level of residual ligase activity. The polymorphisms decrease the activity of DNA ligase IV by approximately 2-fold. When combined with the otherwise mild R278H mutation, the activity is reduced to a level similar to other LIG4 patients who display immunodeficiency and developmental delay. This demonstrates how coupling of a mutation and polymorphism can have a marked impact on protein function and provides an example where a polymorphism may have influenced clinical outcome. Analysis of additional mutational changes in LIG4 syndrome (R580X, R814X and G469E) have led to the identification of a nuclear localization signal in DNA ligase IV and sites impacting upon DNA ligase IV adenylation.     

Expression of the human T cell receptor V beta repertoire.

We have used a sensitive assay, based on amplification of cDNA by the polymerase chain reaction, to determine in a variety of human tissues the relative levels of expression of the genes coding for each of the twenty families of human TcR V beta. We have determined the diversity of the expressed TcR V beta repertoire early in the development of the immune system. We have shown that the full TcR V beta repertoire is expressed early into the second trimester; the expressed repertoire is as diverse at this point, in both fetal thymus and spleen, as it is in mature thymus and peripheral blood lymphocytes. In addition the relative expression in the fetal thymus of each V beta gene is conserved to a large extent in the fetal spleen.     

The effect of cromolyn sodium on hypoxia-induced histamine release.

In 16 guinea pigs the pulmonary circulation was isolated from the pulmonary systemic circulation and perfused with Tris buffer or with Tris buffer containing cromolyn sodium. The lungs were first ventilated with 100% O2 and then 100% N2 to induce alveolar hypoxia. The effluent was collected from the aorta and assayed for histamine by the fluorometric method. In all seven animals perfused with Tris alone (no cromolyn) a marked elevation of the effluent histamine was demonstrated. In the group perfused with buffer containing cromolyn there was a substantial diminution in the response to hypoxia. Therefore, cromolyn sodium partially blocked hypoxia-mediated histamine release.     

Changing patterns of dominance in the CD8+ T cell response during acute and persistent murine gamma-herpesvirus infection

The murine gamma-herpesvirus MHV-68 causes an acute, transient pneumonitis, followed by an infectious mononucleosis (IM)-like illness with splenomegaly, widespread latent infection of B lymphocytes and an expansion of Vbeta4+ CD8+ T cells. CD8+ T cells specific for an H-2Db-restricted epitope were prominent during the acute respiratory infection, but their prevalence declined rapidly during the mononucleosis. In contrast, CD8+ T cells specific for an H-2Kb-restricted epitope, apparently expressed by virus-infected B lymphocytes, were most numerous during the mononucleosis illness and were maintained at relatively high frequencies thereafter. The prevalence of all peptide-specific CD8+ T cells decreased during the expansion of the Vbeta4+ CD8+ population, which did not recognize any peptide epitopes identified and was apparent also in an MHC class I-deficient environment. The CD8+ T cell population recognizing productively infected epithelial cells thus differed substantially from that responding during the IM illness.     

Current Biotechnological Approaches to the Prevention of Restenosis

No systemic pharmacological treatment has been convincingly shown to reduce the incidence of restenosis after angioplasty in patients. The lack of success of many pharmaceutical agents in reducing restenosis rates post-angioplasty and following stent implantation, as documented in dozens of clinical trials, has encouraged the development of new biotechnological approaches to the treatment of restenosis. Gene therapy and other agents, including antibodies, fusion toxins and ribozymes, have the potential to prevent some of the sequelae after arterial injury, particularly cell proliferation. Mechanical methods of preventing restenosis, for example sophisticated local drug delivery strategies and biodegradable stents using new materials, in combination with novel therapeutic agents or radiation, may also be of use.     

Solubility properties in polymers and biological media. 2. The correlation and prediction of the solubilities of nonelectrolytes in biological tissues and fluids

Solubilities of a range of nonelectrolyte solutes in biological systems, such as blood, plasma, brain, lung, liver, kidney, muscle tissue, and human fat, are correlated and predicted through an equation that takes the form log Ltissue = c + w log Lwater + o log Loil, where L is the Ostwald solubility coefficient (or gas/liquid partition coefficient). The ratio of the constants o and w gives a measure of the "oiliness" of a given biological tissue or fluid. The strong possibility exists that, for many types of nonelectrolyte solutes, simple measurements of solubilities in water and oil (gas/liquid partition coefficients) will allow accurate predictions of solubilities in the above biological solvents, as well as tissue/blood partition coefficients. The solubility of rare gases and the inorganic gases H2, N2, CO, and O2 may be correlated through the simpler equation log Ltissue = l'RG + d', where l' and d' are constants that characterize the phase, and RG is a known parameter, obtained by normalizing and averaging solubilities over a range of solvent systems, that characterizes the solute. Both of the above equations allow prediction of L in biological solvents to within about 20%, which compares well with the precision of the experimental measurements.     

Evidence for solid- and liquid-state interactions in a furosemide-polyvinylpyrrolidone solid dispersion

The solid-state interactions between furosemide (FUR) and polyvinylpyrrolidone (PVP) in a solid dispersion system have been investigated by dispersive and Fourier Transform IR. Using spectral subtraction procedures, shifts to lower frequencies are seen in FUR-NH stretching vibrations in amorphous FUR-PVP systems in comparison with the crystalline FUR spectrum. The magnitude of shifts are similar to those seen when FUR is dissolved in the hydrogen-bonding solvent 1,4 dioxan (0.015-0.155 M, 20 degrees C). Spectral assignment indicates the FUR sulfonamide group binds to PVP in preference to the FUR secondary amine which is shown to possess an intramolecular hydrogen bond by analysis of published X-ray crystal data. With a molecular graphics computer program, the FUR crystal unit cell was compiled, and analyses of nonbonded intermolecular distances are useful in interpreting IR shift data in amorphous solid dispersions. A study of FUR-PVP interactions in solution, examined by proton NMR, reveals a downfield shift of the FUR sulfonamide proton resonance. This shift exhibits dilution and temperature (298-338 K) dependence consistent with an intermolecular hydrogen bond. The secondary amine proton resonance exhibits temperature but not dilution or solvent bonding potential dependence, characteristics of an intramolecular hydrogen bond. The proposed hydrogen bond FUR-PVP interaction may account for the formation and stabilization of the amorphous solid dispersion.     

What do patients really want? Redefining a behavioral science curriculum for family physicians

This study replicates the design reported by Schwenk et al and addresses a key methodologic issue in their paper. The original questionnaire by Schwenk et al was administered to one half of the sample of patients, while the other half completed a reworded questionnaire asking what they "want" in the area of psychosocial help, as opposed to what they think their family physician "would" do (the original wording). The hypothesis was that expectations for physician involvement will be higher if patients are asked what they want as opposed to what they expect. Patients were asked to complete a four-page questionnaire, alternating the questions described by Schwenk et al with the reworded questionnaire, in which they were required to rank the level of involvement requested from their physician regarding 45 psychosocial problems (level 1 = no involvement, level 4 = major involvement). Results using the originally worded questionnaire closely paralleled findings of Schwenk et al, whereas asking people what they "wanted" showed statistically significant differences in 18 of the 45 items. The paper concludes with discussion of patient preferences vs patient expectations, with implications for the behavioral science curriculum.