Contribution of lipids to the pathogenesis of transplant vascular sclerosis.
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Animals↗
Biomedical subjects
J E Wilson
Publications and source records attributed to J E Wilson.
Human leukocyte antigen class II associations in patients with idiopathic dilated cardiomyopathy. Myocarditis Treatment Trial Investigators.
BACKGROUND: Idiopathic dilated cardiomyopathy (IDC) is a disease of unknown etiology for which immune abnormalities, possibly related to viral infections, are suspected but unproven. Previous serologic studies have reported associations between human leukocyte antigen DR4 and IDC. A molecular study of human leukocyte antigen associations was undertaken in patients with IDC to further explore the possibility of susceptibility markers of genetically determined disease. METHODS AND RESULTS: In this study, 36 patients from the Myocarditis Treatment Trial (32 IDC and 4 myocarditis patients) were examined using restriction fragment length polymorphism analysis and polymerase chain reaction amplification with sequence-specific primers to perform class II typing. All 4 myocarditis patients were DQ5 positive and 3 possessed the allele DQB1*0501. In the IDC group, the frequency of human leukocyte antigen DR4 was similar to that reported in the normal population. In addition, there was no excess prevalence of any molecularly defined DR4 alleles (0401-0419). There was an increase in the frequency of DR12 in IDC patients. The frequencies of the alleles DQB1 *0503 and DQB1*0301 and/or *0304 were also increased in IDC patients versus the normal population. CONCLUSION: The molecular studies point to a relationship between the DQ locus and IDC.
Adult↗
Homologous and heterologous interactions between hexokinase and mitochondrial porin: evolutionary implications.
Binding of the Type I isozyme of mammalian hexokinase to mitochondria is mediated by the porin present in the outer mitochondrial membrane. Type I hexokinase from rat brain is avidly bound by rat liver mitochondria while, under the same conditions, there is no significant binding to mitochondria from S. cerevisiae. Previously published work demonstrates the lack of significant interaction of yeast hexokinase with mitochondria from either liver or yeast. Thus, structural features required for the interaction of porin and hexokinase must have emerged during evolution of the mammalian forms of these proteins. If these structural features serve no functional role other than facilitating this interaction of hexokinase with mitochondria, it seems likely that they evolved in synchrony since operation of selective pressures on the hexokinase-mitochondrial interaction would require the simultaneous presence of hexokinase and porin capable of at least minimal interaction, and be responsive to changes in either partner that affected this interaction. Recent studies have indicated that a second type of binding site, which may or may not involve porin, is present on mammalian mitochondria. There are also reports of hexokinase binding to mitochondria in plant tissues, but the nature of the binding site remains undefined.
Animals↗
Conducting ecologically valid prevention research: recruiting and retaining a "whole village" in multimethod, multiagent studies.
Many prevention studies are now designed with complementary interventions in different settings. Evaluations of these interventions require assessing the child's behavior in each of these settings. Conducting these studies, therefore, may involve recruiting school districts, principals, classroom teachers, peers, parents, siblings, and in later years, employers and intimate partners. These participants may be considered natural raters or satellite subjects, depending on their degree of involvement. Issues of recruitment and retention thus are magnified in multimethod, multiagent studies. To illustrate these issues, findings are presented for three studies conducted with risk populations in the past decade at the Oregon Social Learning Center: a passive longitudinal study, a selected prevention study, and an indicated prevention study. Findings indicate that achieving high recruitment and retention rates for at-risk and high-risk subjects in multisetting studies is possible, and that a developmental approach should be taken to recruiting risk populations.
Adolescent↗
An introduction to the isoenzymes of mammalian hexokinase types I-III.
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Animals↗
Inhibitors of protein kinases and phosphatases alter root morphology and disorganize cortical microtubules.
To investigate molecular mechanisms controlling plant morphogenesis, we examined the morphology of primary roots of Arabidopsis thaliana and the organization of cortical microtubules in response to inhibitors of serine/threonine protein phosphatases and kinases. We found that cantharidin, an inhibitor of types 1 and 2A protein phosphatases, as previously reported for okadaic acid and calyculin A (R.D. Smith, J.E. Wilson, J.C. Walker, T.I. Baskin [1994] Planta 194: 516-524), inhibited elongation and stimulated radial expansion. Of the protein kinase inhibitors tested, chelerythrine, 6-dimethylaminopurine, H-89, K252a, ML-9, and staurosporine all inhibited elongation, but only staurosporine appreciably stimulated radial expansion. To determine the basis for the root swelling, we examined cortical microtubules in semithin sections of material embedded in butyl-methyl-methacrylate. Chelerythrine and 100 nM okadaic acid, which inhibited elongation without causing swelling, did not change the appearance of cortical arrays, but calyculin A, cantharidin, and staurosporine, which caused swelling, disorganized cortical microtubules. The stability of the microtubules in the aberrant arrays was not detectably different from those in control arrays, as judged by similar sensitivity to depolymerization by cold or oryzalin. These results identify protein phosphorylation and dephosphorylation as requirements in one or more steps that organize the cortical array of microtubules.
Arabidopsis↗
Myocarditis as systemic disease: new perspectives on pathogenesis.
1. Myocarditis may be an early indicator of or may subsequently lead to dilated cardiomyopathy in humans. This hypothesis has evolved from research on viruses that induce myocarditis, wherein the coxsackie B group viruses (CVB) in the family Picornaviridae are the most common known viral infectants of heart muscle. 2. Many competing hypotheses exist as to the pathogenesis of CVB3-induced myocarditis, including direct virus-induced myocyte damage and immunopathological disease with autoimmune sequelae. Evidence to support the direct-damage and viral RNA-persistence hypothesis is derived from in situ hybridization and gene amplification studies. 3. Recent use of terminal deoxynucleotidyl transferase-mediated nick-end labelling indicates that this injury in target organs is largely non-apoptotic in nature. Most apoptotic bodies in cardiac tissue are derived from immune cells. 4. Beyond infection of heart muscle, CVB3 can also associate with, infect and persist in cells of immune origin. The CVB3 localizes to follicles in spleens and lymph nodes of the murine host and this particular localization may continue in mice susceptible to more aggressive myocarditis. Whether virus-immune cell association in these compartments is advantageous (or essential) to the host in the evolution of anti-viral immune responses or whether it is more advantageous to the virus in immunosuppression of the host is not known. 5. We suggest that CVB3 can directly perturb or alter the immune response, thereby delaying viral clearance from vulnerable systemic organs. Both host and viral genetic factors can influence susceptibility, persistence and disease progression. 6. Picornaviruses use a unique method for the initiation of translation, involving the internal binding of the ribosome on a sequence element of the 5' untranslated region, termed an internal ribosome entry site (IRES). 7. The IRES of CVB3 is located at approximately stem loops G, H and I, spanning nucloetides 530 and 630. Arrest of host translation is also a feature of picornavirus infection. Such regulation of host cell translation machinery no doubt fosters viral replication at the expense of the host cell. 8. Differences between cell types in the mechanisms, along with those at other key steps in the viral life cycle and in signalling via kinase pathways, may determine viral tropism and cellular destruction and the physiological outcome of neighbouring cells.
Animals↗
Complement component 3 interactions with coxsackievirus B3 capsid proteins: innate immunity and the rapid formation of splenic antiviral germinal centers.
Innate immunity is central to the clearance of pathogens from hosts as well as to the definition of acquired immune responses (D. T. Fearon, and R. M. Locksley, Science 272:50-53, 1996). Coxsackievirus B3 (CVB3), a human cardiopathic virus, was evaluated for the ability to activate the alternative and classical pathway of complement. CVB3 proteins interact with complement component 3 (C3, a soluble protein effector of innate immunity) after either in vitro exposure to mouse serum or in vivo murine infection and activate the alternative pathway of complement. In addition, we demonstrate that viral antigen retention and localization in germinal centers is dependent on C3, while virus antigen retention in extrafollicular regions in the spleen is not. In vivo depletion of native C3 abolished the rapid formation of virus-specific germinal centers (by day 3 post-CVB3 infection) in the absence of serum anti-CVB3 antibodies. These studies demonstrate that innate immune mechanisms, such as C3 interaction with CVB3, are essential for splenic antiviral germinal center formation in naive (antigen nonsensitized) mice resistant (C57BL/6J strain) and susceptible (A/J strain) to CVB3-induced myocarditis.
Animals↗
Possible association of a reduction in cardiovascular events with blood donation.
BACKGROUND: The iron hypothesis suggests that females are protected from atherosclerosis by having lower iron stores than men, thus limiting oxidation of lipids. OBJECTIVE: To test the iron hypothesis by comparing cardiovascular event rates in whole blood donors compared with nondonors. DESIGN: Prospective cohort with telephone survey follow up. SETTING: The State of Nebraska, USA. PARTICIPANTS: A sample was selected from the Nebraska Diet Heart Survey (NDHS) restricting for age > or = 40 years and absence of clinically apparent vascular diseases at time of enrollment in to NDHS (1985-87). MAIN OUTCOME MEASURES: The occurrence of cardiovascular events (myocardial infarction, angina, stroke), procedures (angioplasty, bypass surgery, claudication, endarterectomy), nitroglycerin use, or death (all cause mortality), and level of blood donation. RESULTS: Participants were 655 blood donors and 3200 non-donors who differed in education, physical activity, diabetes, and frequency of antihypertensive treatment; 889 were lost to follow up. Sixty four donors and 567 non-donors reported cardiovascular events (crude odds ratio = 0.50, 95% confidence interval (CI) 0.38-0.66). The benefit of donation was confined to non-smoking males (adjusted odds ratio 0.67, 95% CI 0.45-0.99). Benefit was limited to current donors (the most recent three years). No additional benefit resulted from donating more than once or twice over three years. CONCLUSION: In support of the iron hypothesis, blood donation in non-smoking men in this cohort was associated with reduced risk of cardiovascular events. A randomised clinical trial is warranted to confirm these findings as the observed personal health benefit of donation has public policy ramifications.
Adolescent↗
Correction of hypertriglyceridemia and impaired fat tolerance in lipoprotein lipase-deficient mice by adenovirus-mediated expression of human lipoprotein lipase.
Humans homozygous or heterozygous for mutations in the lipoprotein lipase (LPL) gene demonstrate significant disturbances in plasma lipoproteins, including raised triglyceride (TG) and reduced HDL cholesterol levels. In this study we explored the feasibility of adenovirus-mediated gene replacement therapy for LPL deficiency. A total of 5 x 10(9) plaque-forming units (pfu) of an E1/E3-deleted adenovirus expressing either human LPL (Ad-LPL) or the bacterial beta-galactosidase gene (Ad-LacZ) as a control were administered to mice heterozygous for targeted disruption in the LPL gene (n = 57). Peak expression of total postheparin plasma LPL activity was observed at day 7 in Ad-LPL mice versus Ad-LacZ controls (834 +/- 133 vs 313 +/- 89 mU/mL, P < .01), and correlated with human-specific LPL activity (522 +/- 219 mU/mL) and mass (9214 +/- 782 ng/mL), a change that was significant to 14 and 42 days, respectively. At day 7, plasma TGs were significantly reduced relative to Ad-LacZ mice (0.17 +/- 0.07 vs 1.90 +/- 0.89 mmol/L, P < .01) but returned to endogenous levels by day 42. Ectopic liver expression of human LPL was confirmed by in situ hybridization analysis and from raised LPL activity and mass in liver homogenates. Analysis of plasma lipoprotein composition revealed a marked decrease in VLDL-derived TGs. Severely impaired oral and intravenous fat-load tolerance in LPL-deficient mice was subsequently corrected after Ad-LPL administration and closely paralleled that observed in wild-type mice. These findings suggest that liver-targeted adenovirus-mediated LPL gene transfer offers an effective means for transient correction of altered lipoprotein metabolism and impaired fat tolerance due to LPL deficiency.
Adenoviridae↗
Enhanced lymphocyte longevity and absence of proliferation and lymphocyte apoptosis in Quilty effects of human heart allografts.
"Quilty effect" (QE) is a common and problematic observation in endomyocardial biopsy specimens from patients after cardiac transplantation. The origin, fate, and significance of QE cellular elements are unknown. Twenty-six paraffin-embedded endomyocardial biopsy specimens with QE (five QE As and twenty-one QE Bs) from twenty-two cardiac allografts were studied by immunohistochemistry for expression of Bcl-2, Fas antigen, proliferating cell nuclear antigen (PCNA), perforin, T cells (UCHL-1), macrophages (CD68), and apoptosis by in situ terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL). Approximately 50% of the lymphocytes present, mainly in the deeper region of 20 of 21 QE Bs and all 5 QE As, expressed Bcl-2 in a pseudo-nodular pattern surrounding high endothelial venules. Fas expression was detected in lymphocytes in 20 of 21 QE Bs and 5 QE As in a similar pattern to Bcl-2. However, endothelial cells and macrophages were Bcl-2 negative, whereas both cell types were Fas positive. Perforin was negative in nearly all lymphocytes. TUNEL staining revealed that lymphocytes in QEs did not undergo apoptosis; however, TUNEL positivity was observed in approximately 70% of endothelial cells and macrophages and certain adjacent cardiac myocytes in 20 of 21 QE Bs and 5 QE As. One large QE B with a germinal center was noted. Germinal center cells expressed PCNA intensely but were negative for Bcl-2, Fas, and TUNEL. Cells surrounding the germinal center expressed abundant Bcl-2. The following conclusions were drawn. 1) Apoptosis does not occur in lymphocytes in QE where enhanced Bcl-2 (apoptosis inhibitor) and Fas antigen (apoptosis inducer) are expressed. 2) PCNA negativity indicates that QE lymphocytes may not proliferate, and perforin negativity indicates that they may not exhibit perforin-based cytotoxicity. We propose that there may be a relationship between the longevity of lymphocytes in QE and the absence of apoptosis.
Adult↗
Diet-induced atherosclerosis in the domestic cat.
The domestic cat has not been used in studies of atherosclerosis, with the exception of a single study published in 1970. We have further evaluated the susceptibility of the domestic cat to diet-induced atherosclerosis, the ultimate intent being to discern the atherogenic risk due to lipoprotein lipase deficiency in an affected feline kindred with a phenotype very similar to that of the human form of this condition. We subjected a group of normal domestic cats to a moderately high-fat, cholesterol-enriched diet (30% fat and 3% cholesterol) for a period of 2 to 8 months. Plasma lipid levels were monitored monthly. At the time of killing, all organs and the entire vascular tree were removed, sectioned, processed, and stained with hematoxylin and eosin. The entire vascular tree was also stained with Movat's pentachrome and oil red O (ORO) and assessed semiquantitatively (0 to 5+/5+) and quantitatively (mean intimal area and ORO positivity, mm2). Both blood lipid measurements (total cholesterol, high-density lipoprotein-cholesterol, triglycerides, and low-density lipoprotein-cholesterol) and vessel wall lesion assessment (intimal area, mm2) were statistically elevated (p < 0.05) in the cholesterol-fed cats as compared to those on a normal diet. The highest correlations obtained between blood lipid components and vessel wall measures were the percent increase in triglyceride from base line versus the ORO measurement or foam cell grade (r = 0.86), and percent increase in triglycerides versus the intimal area in the lower abdominal aorta (r = 0.91). Similar relationships were found when the intimal area in the brachiocephalic/subclavian vessels was correlated with the absolute triglyceride values (r = 0.85) or with the percent increase in triglycerides (r = 0.83). Thus, we produced atherosclerotic lesions in the cat within 2 to 4 months on a cholesterol-enriched diet; blood lipid levels were highly correlated with lesional measurements in the vessel wall. This study will provide the basis for evaluation of the susceptibility of New Zealand lipoprotein lipase-deficient cats to diet-induced atherosclerosis.
Animal Feed↗
Isolation of the promoter for Type I hexokinase from rat.
A genomic clone containing sequence identical to the 5' region of the cDNA for rat Type I hexokinase was isolated from a lambda Charon 4A library. A 5.4-kb EcoRI fragment from this clone, containing the matching sequence, was sequenced in its entirety. Rapid amplification of 5' cDNA ends (5' RACE), reverse transcription polymerase chain reaction, and ribonuclease protection experiments were consistent with the existence of multiple transcriptional start sites clustered in three regions approximately 460, 300, and 100 nucleotides upstream from the translational start codon. Together with results of previous work, the 5' untranslated sequence defined in the present study accounts for the 4.3-kb mRNA for Type I hexokinase seen on Northern blots. Fragments from the 5' flanking region were cloned into a reporter vector containing the luciferase coding region. Based on transfection experiments with both PC12 and H9c2 cells, promoter activity was associated with a region lying between nucleotide positions -742 and -516 (with A of the ATG codon at the translational start site defined as +1). The promoter region lacks a TATA sequence and, together with the transcriptional start sites, is located within a GC rich segment (a "CpG island") approximately 1 kb in length. These characteristics have previously been associated with the promoter and transcriptional start sites of genes for "housekeeping enzymes."
Animals↗
smaug protein represses translation of unlocalized nanos mRNA in the Drosophila embryo.
nanos mRNA, which encodes the localized component of the Drosophila posterior body patterning determinant, is normally translated only at the posterior pole of the embryo, where the mRNA is concentrated. Here we identify two similar cis-acting sequences in the nanos mRNA 3' untranslated region that mediate translational repression. These sequences bind an embryonic protein of 135 kD, smaug, and we refer to them as smaug recognition elements (SREs). Analysis of point mutations in the SREs reveals a strong correlation between smaug binding and translational repression; mutants unable to bind smaug in vitro are not repressed translationally in vivo, whereas mutants that do bind smaug remain repressed translationally. These results strongly suggest that smaug acts in translational repression of unlocalized nanos mRNA. Translational repression is essential, as embryos expressing a nanos mRNA with mutated SREs develop with anterior body patterning defects and die, despite correct localization of the RNA.
Animals↗
Human immunodeficiency virus type-1 reverse transcriptase. Contribution of Met-184 to binding of nucleoside 5'-triphosphate.
Mutations were made in recombinant human immunodeficiency virus type-1 reverse transcriptase (RT) by substituting methionine 184 with alanine (M184A) or valine (M184V), and steady-state and pre-steady-state kinetic constants were determined. The Km values of M184A RT for dNTPs were larger than those of wt RT for RNA-directed synthesis; the kcat values of M184A RT for processive or distributive synthesis were similar. In contrast to M184A RT, the Km and kcat values of M184V RT for dNTP substrates were similar to those of wt RT. The Ki values of M184V RT for 1-beta-L-nucleoside analogs were increased 30-500-fold relative to wt RT for both RNA- and DNA-directed synthesis. The Kd and kp values of wt RT and M184V RT for dCTP and cis-5-fluoro-1-[2-(hydroxymethyl)-1, 3-oxathiolan-5-yl]cytosine 5'-triphosphate (1-beta-L-FTCTP) were estimated from pre-steady-state kinetics for single nucleotide incorporation. The Kd value of M184V RT for 1-beta-L-FTCTP was 19-fold greater than that of wt RT; the kpvalues of the two enzymes were similar. These results support the hypothesis that methionine 184 in the highly conserved YMDD region of wt RT participates in the binding of the nucleoside (analog) 5'-triphosphate.
Amino Acid Sequence↗
Allograft inflammatory factory-1. A cytokine-responsive macrophage molecule expressed in transplanted human hearts.
Allograft inflammatory factor-1 (AIF-1), a cytokine-responsive macrophage molecule, was originally identified and cloned from rat cardiac allografts with chronic cardiac rejection. We performed the present study to determine whether AIF-1 was also involved in the inflammatory response associated with human cardiac transplant rejection. AIF-1 gene transcripts were identified by the reverse-transcriptase polymerase chain reaction in endomyocardial biopsy specimens from human heart transplants and in macrophage cell lines. In the 441-base pair coding region of the human and rat cDNAs, the nucleotide sequences were 86% identical and the deduced amino acid sequences were 90% identical. Consistent with our studies in the rat. AIF-1 was selectively expressed in human macrophage-like cell lines, and immunostaining in human heart allografts localized the AIF-1 gene product to a subset of CD68+ macrophages in the interstitial and perivascular spaces. The parallels between rat and human AIF-1 expression suggest that AIF-1 may have a common effect on the function of activated macrophages in cardiac allografts.
Animals↗
Functional organization of mammalian hexokinases: both N- and C-terminal halves of the rat type II isozyme possess catalytic sites.
Previous work has shown that catalytic function is associated exclusively with the C-terminal half of the Type I isozyme of mammalian hexokinase. In contrast, we now demonstrate that both halves of the Type II isozyme possess comparable catalytic activities. Mutation of a catalytically important Ser residue to Ala at analogous positions in either the N- or the C-terminal halves (S155A or S603A, respectively) of the rat Type II isozyme resulted in approximately 60% reduction in specific activity of the enzyme, with more than 90% reduction in the doubly mutated enzyme (S155A/S603A). Catalytic activity was retained in a chimeric hexokinase comprising the N-terminal half of Type II hexokinase and catalytically inactive (by site-directed mutation) C-terminal half of the Type I isozyme. The N- and C-terminal catalytic sites of Type II hexokinase are similar in V(max) and K(m) (approximately equal to 130 microM) for glucose; however, the N-terminal site has a lower (0.45 vs 1.1 mM) K(m) for ATP, is slightly more sensitive to inhibition by the product analog 1,5-anhydroglucitol-6-P, and is much more sensitive to inhibition by P(i). It is suggested that the Type II isozyme most closely resembles the 100-kDa hexokinase which resulted from duplication and fusion of a gene encoding an ancestral 50-kDa hexokinase and which was the precursor for the contemporary Type I, Type II, and Type III mammalian isozymes. Subsequent evolutionary changes could then have led to functional differentiation of the N- and C-terminal halves, as seen with the Type I (and possibly the Type III) isozyme.
Animals↗
Recombinant human immunodeficiency virus type 1 reverse transcriptase is heterogeneous.
Recombinant wild type (wt) and T215Y HIV-1 reverse transcriptase (RT) were isolated using three methods designated A, B, and C. The three samples of wt RT were kinetically indistinguishable with respect to dTTP turnover on poly(rA).p(dT)10. However, whereas the kinetic constants for dTTP and AZTTP for both T215Y B and T215Y C were similar to those of wt protein, T215Y A exhibited a twofold increase in Km value for dTTP and a 13-fold increase in Ki value for AZTTP with respect to wt protein purified in the same manner. We further investigated this observation by studying the denaturation of wt RT by urea. The urea denaturation curves monitored by fluorescence and circular dichroism spectroscopy were not coincident with the denaturation curve monitored by enzyme activity and yielded Cm values (the concentration of urea at which 50% of the protein is denatured) of 4.1 and 2.0 M urea, respectively. The noncoincidence of the transition curves reflects two separable, sequential, noncooperative conformational changes in the molecule: (a) from a catalytically active to an inactive conformation, and (b) from a catalytically inactive to a denatured, unfolded conformation. We therefore used denaturation as detected by changes in enzyme activity to compare the conformational stability of the three samples of wt and T215Y RT A, B, and C. The Cm values for T215Y RT did not differ from those of the respective wt; however, differences in Cm values were noted depending on how the protein was isolated. This suggested that the heterogeneity of the recombinant RT was due to small differences in conformation at or near the active site.
Antiviral Agents↗