Oncogenicity of DNA in vivo: Tumor induction with expression plasmids for activated H-ras and c-myc

All vaccines and other biological products contain contaminating residual DNA derived from the production cell substrate. Whether this residual cell-substrate DNA can induce tumors in vaccine recipients and thus represent a risk factor has been debated for over 50 years without resolution. As a first step in resolving this issue, we have generated expression plasmids for the activated human H-ras oncogene and for the murine c-myc proto-oncogene. Their oncogenic activity was confirmed in vitro using the focus-formation transformation assay. Two strains of adult and newborn immune-competent mice were inoculated with different amounts of either plasmid alone or with a combination of the H-ras and c-myc plasmids. Tumors developed only in mice inoculated with both plasmids and only at the highest amount of DNA (12.5 microg of each plasmid). The NIH Swiss mouse was more sensitive than the C57BL/6 mouse, and newborn animals were more sensitive than adults. Cell lines were established from the tumors. PCR and Southern hybridization analyses demonstrated that both inoculated oncogenes were present in all of the tumor-derived cell lines and that the cells in the tumors were clonal. Western analysis demonstrated that both oncoproteins were expressed in these cell lines. These results demonstrate that cellular oncogenes can induce tumors following subcutaneous inoculation. Such information provides a possible way of evaluating and estimating the theoretical oncogenic risk posed by residual cell-substrate DNA in vaccines.     

P188 reduces cell death and IGF-I reduces GAG release following single-impact loading of articular cartilage

Prior joint injury predisposes an individual to developing post-traumatic osteoarthritis, for which there is presently no disease modifying treatment. In this condition, articular cartilage degenerates due to cell death and matrix breakdown, resulting in tissue with diminished biomechanical function. P188, a nonionic surfactant, and the growth factor IGF-I have been shown to decrease cell death. Additionally, IGF-I is known to have beneficial effects on cartilage matrix. The objective of this study was to determine the efficacy of P188, IGF-I, and their combination following articular cartilage impact injury with two energy levels, 1.1 J ("low") and 2.8 J ("high"), at 24 h and 1 week. Bovine articular cartilage with attached underlying bone was impacted at the low or high level. Impact sites were explanted and examined immediately, or cultured for 24 h or 1 week in serum-free media supplemented with P188 (8 mgml), IGF-I (100 ngml), or their combination. Gross morphology, cell viability, GAG release to the media, and tissue mechanical properties were assessed. Immediately postimpact, high level impacted tissue had significantly increased gross morphology scores, indicating tissue damage, which were maintained over 1 week. Gross scores following low impact were initially similar to nonimpacted controls, but, at 24 h and 1 week, low impact gross scores significantly increased compared to nonimpacted controls. Additionally, at 24 h, high impact resulted in increased cell death, and both low and high impacts had increased GAG release compared to nonimpacted controls. Furthermore, high impact caused decreased tissue stiffness at 24 h that appeared to worsen over 1 week, evident by the percent decrease from nonimpacted controls increasing from 16% to 26%. No treatment type studied mitigated this loss. The combination did not perform better than either individual treatment; however, following low impact at 1 week, P188 reduced cell death by 75% compared to no treatment and IGF-I decreased GAG release from the tissue by 49%. In conclusion, high impact resulted in immediate tissue changes that worsened over 1 week. Though not causing immediate changes, low impact also resulted in tissue degeneration evident by 24 h. No treatment studied was effective at 24 h, but by 1 week P188 and IGF-I ameliorated established detrimental changes occurring in articular cartilage postimpact. However, further work is needed to optimize treatment strategies to prevent and/or reverse cell death and matrix destruction in a way that maintains tissue mechanical properties, and hence its functionality.     

The Mutational Spectrum of Lynch Syndrome in Cyprus

Lynch syndrome is the most common form of hereditary colorectal cancer and is caused by germline mutations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. Mutation carriers have an increased lifetime risk of developing colorectal cancer as well as other extracolonic tumours. The aim of the current study was to evaluate the frequency and distribution of mutations in the MLH1, MSH2 and MSH6 genes within a cohort of Cypriot families that fulfilled the revised Bethesda guidelines. The study cohort included 77 patients who fulfilled at least one of the revised Bethesda guidelines. Mutational analysis revealed the presence of 4 pathogenic mutations, 3 in the MLH1 gene and 1 in the MSH2 gene, in 5 unrelated individuals. It is noted that out of the 4 pathogenic mutations detected, one is novel (c.1610delG in exon 14 of the MLH1) and has been detected for the first time in the Cypriot population. Overall, the pathogenic mutation detection rate in our patient cohort was 7%. This percentage is relatively low but could be explained by the fact that the sole criterion for genetic screening was compliance to the revised Bethesda guidelines. Larger numbers of Lynch syndrome families and screening of the two additional predisposition genes, PMS2 and EPCAM, are needed in order to decipher the full spectrum of mutations associated with Lynch syndrome predisposition in Cyprus.     

Further studies of the binding specificity of the leukocyte adhesion molecule, L-selectin, towards sulphated oligosaccharides--suggestion of a link between the selectin- and the integrin-mediated lymphocyte adhesion systems

This communication is concerned with the binding specficityof the leukocyte-adhesion molecule L-selectin (leukocyte homingreceptor) towards structurally defined sulphated oligosaccharidesof the blood group Le^a and Le^x series, and of the glycolsaminoglycanseries heparin, chondroitin sulphate and keratan sulphate. Therecombinant soluble form of the rat L-selectin (L-selectin-IgGFc chimera) investigated here was shown previously to bind tolipid-linked oligosaccharides 3-O, 4-O and 6-O sulphated atgalactose, such as sulphatides and a mixture of 3-sulphatedLe^a/Le^x type tetrasaccharides isolated from ovarian cystadenoma,as well as to the HNK-1 glycolipid with 3-O sulphated glucuronicacid. In the present study, the L-selectin investigated in bothchromatogram binding and plastic microwell binding experimentsusing neoglycolipids was found to bind to the individual 3-sulphatedLe^a and Le^x sequences (penta-, tetra- and trisaccharides), andwith somewhat lower intensities to their non-fucosylated analogues.Glycosaminoglycan disaccharides of keratan sulphate, heparinand chondroitin sulphate types were also bound by L-selectinin one or both assay systems, leading to the conclusion thatclustered glycosaminoglycan oligosaccharides with 6-O sulphationof N-acetylgalactctosamine, N-acetylglucosamine or glucosamine,4-O sulphation of N-acetylgalactosamine, 2-O sulphation of uronicacid, N-sulphation of glucosamine and, to a lesser extent, thenon-sulphated uronic acid-contahing disaccharides, can supportL-selectin adhesion. As inflammatory chemokines (short-rangestimulators of lymphocyte migration which trigger integrin activation)are known to bind to endothelial glycosaminoglycans, we proposethat the binding of the lymphocyte membrane L-selectin to endothelialglycosaminoglycans may provide a link between the selectin-mediatedand integrin-mediated adhesion systems in leukocyte extravasationcascades. The posibility is also raised that lymphocyte L-selectininteractions with glycosaminoglycans may contribute to pathologiesof glycosaminoglycan-rich tissues, e.g. cartilage loss in rheumatoidarthritis and inflammatory lesions of the cornea. glycosaminoglycans leukocyte adhesion cascades neoglycolipids oligosaccharide presentation sulphated oligosaccharides     

Carbocyclic thromboxane A2: Aggrevation of myocardial ischemia by a new synthetic thromboxane A2 analog

$\text{In vitro}$ experiments indicate that thromboxane A₂ (TA₂) is a potent platelet aggregator and vascular constrictor. However, it is unclear what roles these specific actions may contribute in the pathophysiology of myocardial ischemia. Carbocyclic thromboxane A₂ (CTA₂), a TA₂ analog, constricts isolated perfused cat coronary arteries, but does not aggregate platelets, and thus appeared useful to clarify these separate actions of TA₂. In anesthetized cats, radioactive labeled microspheres were injected into the left atrium for measurement of cardiac output and tissue blood flows. Compared to control measurements, CTA₂ infusion (4.8 μg·kg^?1·min^?1 to 10 min) significantly decreased cardiac output from 347 ± 16 ml·min^?1 to 248 ± 16 ml·min^?1 (p<0.025). Furthermore, V7 CTA₂ also significantly reduced blood flow to the left ventricle by 33 ± 7%, but did not alter heart rate or MABP in the intact cat. In cats subjected to left anterior descending coronary artery occlusion, infusion of CTA₂ (1 μg·min^?1 for 120 minutes) 30 min after ligation resulted in a significantly reduced myocardial cellular integrity as measured by myocardial creatine kinase activity (p<0.01) or percent bound myocardial cathepsin D (p<0.01). Thus, these data suggest that activation of vascular thromboxane receptors as well as direct cellular damage may play a role in the pathophysiology of myocardial ischemia.     

Morph-Specific Proteins in Pollen and Styles of Distylous Turnera (Turneraceae)

We used nondenaturing isoelectric focusing (IEF) in a survey of plants from 11 populations to identify style and pollen proteins unique to the short-styled morph of Turnera scabra, T. subulata and T. krapovickasii. Three protein bands [approximately isoelectric points (pIs) 6.1, 6.3 and 6.5] were found only in styles and stigmas of short-styled plants while two bands (approximately pIs 6.7 and 6.8, M(r) 56 and 59 kD) occur only in pollen of short-styled plants. Some of these bands appear very late in development, within 24 hr before flowering. Two isozyme loci were mapped to an 8.7 cM region spanning the distyly locus. Using these isozyme markers we identified progeny exhibiting recombination adjacent to the distyly locus. No recombinants between the distyly locus and the locus or loci controlling the presence of the short-styled morph-specific proteins were obtained. This suggests that the loci encoding these proteins are either extremely tightly linked to the distyly locus and in complete disequilibrium with the S allele or exhibit morph-limited expression. Crosses to a plant showing an unusual style protein phenotype demonstrated that an additional unlinked locus is required for full expression of the style proteins. The function of the morph-specific proteins is unknown.     

A model of growing vascular structures

Increasing attention is being paid to the configuration and development of vascular structures and their possible correlations with physiological events. The study of angiogenesis in normal and pathological states as well as in the embryo and adult has provided new insights into the mechanism of vessel growth and organization of the vasculature. Various mathematical branching models have been developed. These constructions are mainly geometrical and only involve a branching phenomenon. We propose the use of a deterministic non-linear model based on physiological laws and hydrodynamics. Growth, branching and anastomosis, the three actual main events occurring in vascular growth, are included in this model. Space growth, including cells and vessels, is defined by a decreasing transformation. Space density and the length of new sprouts are controlled by a set of parameters. The conditions on these parameters are well established, which allows the production of realistic patterns.     

Synthesis and vascular actions of an arachidonic acid ethylene-diamino-triethyl-ester (AA-EDTA) derivative

An ethylene-diamino-triethyl-ester derivative of arachidonic acid (AA-EDTA) was newly synthetized and tested for its coronary vasoactivity in isolated perfused cat coronary arteries. This arachidonic acid analog exerted a coronary vasodilator effect and significantly antagonized the coronary vasoconstrictor effect of LTD₄. The constrictor response to the thromboxane analog carbocyclic thromboxane A₂ was unaffected by AA-EDTA. These properties of AA-EDTA may be useful in counteracting the vasoconstrictor influence of leukotrienes in situations such as coronary artery vasospasm.     

Molecular Modeling of Mechanosensory Ion Channel Structural and Functional Features

The DEG/ENaC (Degenerin/Epithelial Sodium Channel) protein family comprises related ion channel subunits from all metazoans, including humans. Members of this protein family play roles in several important biological processes such as transduction of mechanical stimuli, sodium re-absorption and blood pressure regulation. Several blocks of amino acid sequence are conserved in DEG/ENaC proteins, but structure/function relations in this channel class are poorly understood. Given the considerable experimental limitations associated with the crystallization of integral membrane proteins, knowledge-based modeling is often the only route towards obtaining reliable structural information. To gain insight into the structural characteristics of DEG/ENaC ion channels, we derived three-dimensional models of MEC-4 and UNC-8, based on the available crystal structures of ASIC1 (Acid Sensing Ion Channel 1). MEC-4 and UNC-8 are two DEG/ENaC family members involved in mechanosensation and proprioception respectively, in the nematode Caenorhabditis elegans. We used these models to examine the structural effects of specific mutations that alter channel function in vivo. The trimeric MEC-4 model provides insight into the mechanism by which gain-of-function mutations cause structural alterations that result in increased channel permeability, which trigger cell degeneration. Our analysis provides an introductory framework to further investigate the multimeric organization of the DEG/ENaC ion channel complex.     

Strain-dependent Recovery Behavior of Single Chondrocytes

One of the challenges facing researchers studying chondrocyte mechanobiology is determining the range of mechanical forces pertinent to the problems they study. One possible way to deal with this problem is to quantify how the biomechanical behavior of cells varies in response to changing mechanical forces. In this study, the compressibility and recovery behaviors of single chondrocytes were determined as a function of compressive strains from 6 to 63%. Bovine articular chondrocytes from the middle and deep zones were subjected to this range of strains, and digital videocapture was used to track changes in cell dimensions during and after compression. The normalized volume change, apparent Poisson’s ratio, residual strain after recovery, cell volume fraction after recovery, and characteristic recovery time constant were analyzed with respect to axial strain. Normalized volume change varied as a function of strain, demonstrating that chondrocytes exhibited compressibility. The mean Poisson’s ratio of chondrocytes was found to be 0.29 ± 0.14, and did not vary with axial strain. In contrast, residual strain, recovered volume fraction, and recovery time constant all depended on axial strain. The dependence of residual strain and recovered volume fraction on axial strain showed a change in behavior around 25–30% strain, opening up the possibility that this range of strains represents a critical value for chondrocytes. Quantifying the mechanical behavior of cells as a function of stress and strain is a potentially useful approach for identifying levels of mechanical stimulation that may be germane to normal cartilage physiology, functional tissue engineering of cartilage, and the etiopathogenesis of osteoarthritis.