Circular RNAs: Biogenesis, Functions, and Role in Myocardial Hypertrophy.

Circular RNAs (circRNAs) are a large class of endogenous single-stranded covalently closed RNA molecules. High-throughput RNA sequencing and bioinformatic algorithms have identified thousands of eukaryotic circRNAs characterized by high stability and tissue-specific expression pattern. Recent studies have shown that circRNAs play an important role in the regulation of physiological processes in the norm and in various diseases, including cardiovascular disorders. The review presents current concepts of circRNA biogenesis, structural features, and biological functions, describes the methods of circRNA analysis, and summarizes the results of studies on the role of circRNAs in the pathogenesis of hypertrophic cardiomyopathy, the most common inherited heart disease.

Multiplex Analysis of Serum Cytokine Profiles in Systemic Lupus Erythematosus and Multiple Sclerosis.

Changes in cytokine profiles and cytokine networks are known to be a hallmark of autoimmune diseases, including systemic lupus erythematosus (SLE) and multiple sclerosis (MS). However, cytokine profiles research studies are usually based on the analysis of a small number of cytokines and give conflicting results. In this work, we analyzed cytokine profiles of 41 analytes in patients with SLE and MS compared with healthy donors using multiplex immunoassay. The SLE group included treated patients, while the MS patients were drug-free. Levels of 11 cytokines, IL-1b, IL-1RA, IL-6, IL-9, IL-10, IL-15, MCP-1/CCL2, Fractalkine/CX3CL1, MIP-1a/CCL3, MIP-1b/CCL4, and TNFa, were increased, but sCD40L, PDGF-AA, and MDC/CCL22 levels were decreased in SLE patients. Thus, changes in the cytokine profile in SLE have been associated with the dysregulation of interleukins, TNF superfamily members, and chemokines. In the case of MS, levels of 10 cytokines, sCD40L, CCL2, CCL3, CCL22, PDGF-AA, PDGF-AB/BB, EGF, IL-8, TGF-a, and VEGF, decreased significantly compared to the control group. Therefore, cytokine network dysregulation in MS is characterized by abnormal levels of growth factors and chemokines. Cross-disorder analysis of cytokine levels in MS and SLE showed significant differences between 22 cytokines. Protein interaction network analysis showed that all significantly altered cytokines in both SLE and MS are functionally interconnected. Thus, MS and SLE may be associated with impaired functional relationships in the cytokine network. A cytokine correlation networks analysis revealed changes in correlation clusters in SLE and MS. These data expand the understanding of abnormal regulatory interactions in cytokine profiles associated with autoimmune diseases.

Impact of 9p21.3 region and atherosclerosis-related genes' variants on long-term recurrent hard cardiac events after a myocardial infarction.

Atherosclerotic coronary artery disease (CAD) and myocardial infarction (MI) as its most severe clinical complication remain the leading causes of mortality in the majority of countries. Despite the progress in the treatment of MI, quite often the patients, after the first-time MI, develop subsequently a variety of adverse cardiovascular events. In this retrospective study we evaluated the contribution of allelic variations in 9p21.3 locus and in 21 atherogenesis-related genes to the development of hard cardiac events in a cohort of patients of Russian ethnicity after the first acute MI during long-term follow-up (7-10 years). Death from cardiac causes and recurrent nonfatal MI were considered as key clinical outcomes. We have shown the association of rs1333049 and rs10757278 in 9p21.3 and MTHFR rs1801133 with recurrent unfavorable events, the latter was observed in time-dependent manner. Multilocus analysis additionally suggested the influence of carriage of the CRP and ENOS genes variants at the development of subsequent adverse events after MI. The composite model built for prediction of the individual genetic risk of postinfarction hard cardiac events included 9p21.3 rs1333049*GG and MTHFR*TT and was characterized by area under the curve (AUC) = 0.65. Our data show that 9p21.3 locus and MTHFR gene polymorphisms could influence long-term prognosis of recurrent hard cardiac events in patients who underwent the first MI. It is possible that addition of genotyping at such loci to existing clinical scores could improve their predictability.

Genetic risk factors for myocardial infarction more clearly manifest for early age of first onset.

Epidemiological genetics established that heritability in determining the risk of myocardial infarction (MI) is substantially greater when MI occurs early in life. However, the genetic architecture of early-onset and late-onset MI was not compared. We analyzed genotype frequencies of SNPs in/near 20 genes whose protein products are involved in the pathogenesis of atherosclerosis in two groups of Russian patients with MI: the first group included patients with age of first MI onset <60 years (N = 230) and the second group with onset ≥60 years (N = 174). The control group of corresponding ethnicity consisted of 193 unrelated volunteers without cardiovascular diseases (93 individuals were over 60 years). We found that in the group of patients with age of onset <60 years, SNPs FGB rs1800788*T, TGFB1 rs1982073*T/T, ENOS rs2070744*C and CRP rs1130864*T/T were associated with risk of MI, whereas in patients with age of onset ≥60 years, only TGFB1 rs1982073*T/T was associated with risk of MI. Using APSampler software, we found composite markers associated with MI only in patients with early onset: FGB rs1800788*T + TGFB1 rs1982073*T; FGB rs1800788*T + LPL rs328*C + IL4 rs2243250*C; FGB rs1800788*T + ENOS rs2070744*C (Fisher p values of 1.4 × 10-6 to 2.2 × 10-5; the permutation p values of 1.1 × 10-5 to 3.0 × 10-4; ORs = 2.67-2.54). Alleles included in the combinations were associated with MI less significantly and with lower ORs than the combinations themselves. The result showed a substantially greater contribution of the genetic component in the development of MI if it occurs early in life, and demonstrated the usefulness of genetic testing for young people.

Variants of the Coagulation and Inflammation Genes Are Replicably Associated with Myocardial Infarction and Epistatically Interact in Russians.

BACKGROUND: In spite of progress in cardiovascular genetics, data on genetic background of myocardial infarction are still limited and contradictory. This applies as well to the genes involved in inflammation and coagulation processes, which play a crucial role in the disease etiopathogenesis. METHODS AND RESULTS: In this study we found genetic variants of TGFB1, FGB and CRP genes associated with myocardial infarction in discovery and replication groups of Russian descent from the Moscow region and the Republic of Bashkortostan (325/185 and 220/197 samples, correspondingly). We also found and replicated biallelic combinations of TGFB1 with FGB, TGFB1 with CRP and IFNG with PTGS1 genetic variants associated with myocardial infarction providing a detectable cumulative effect. We proposed an original two-component procedure for the analysis of nonlinear (epistatic) interactions between the genes in biallelic combinations and confirmed the epistasis hypothesis for the set of alleles of IFNG with PTGS. The procedure is applicable to any pair of logical variables, e.g. carriage of two sets of alleles. The composite model that included three single gene variants and the epistatic pair has AUC of 0.66 both in discovery and replication groups. CONCLUSIONS: The genetic impact of TGFB1, FGB, CRP, IFNG, and PTGS and/or their biallelic combinations on myocardial infarction was found and replicated in Russians. Evidence of epistatic interactions between IFNG with PTGS genes was obtained both in discovery and replication groups.

Genome-wide significant association with seven novel multiple sclerosis risk loci.

OBJECTIVE: A recent large-scale study in multiple sclerosis (MS) using the ImmunoChip platform reported on 11 loci that showed suggestive genetic association with MS. Additional data in sufficiently sized and independent data sets are needed to assess whether these loci represent genuine MS risk factors. METHODS: The lead SNPs of all 11 loci were genotyped in 10 796 MS cases and 10 793 controls from Germany, Spain, France, the Netherlands, Austria and Russia, that were independent from the previously reported cohorts. Association analyses were performed using logistic regression based on an additive model. Summary effect size estimates were calculated using fixed-effect meta-analysis. RESULTS: Seven of the 11 tested SNPs showed significant association with MS susceptibility in the 21 589 individuals analysed here. Meta-analysis across our and previously published MS case-control data (total sample size n=101 683) revealed novel genome-wide significant association with MS susceptibility (p<5×10(-8)) for all seven variants. This included SNPs in or near LOC100506457 (rs1534422, p=4.03×10(-12)), CD28 (rs6435203, p=1.35×10(-9)), LPP (rs4686953, p=3.35×10(-8)), ETS1 (rs3809006, p=7.74×10(-9)), DLEU1 (rs806349, p=8.14×10(-12)), LPIN3 (rs6072343, p=7.16×10(-12)) and IFNGR2 (rs9808753, p=4.40×10(-10)). Cis expression quantitative locus effects were observed in silico for rs6435203 on CD28 and for rs9808753 on several immunologically relevant genes in the IFNGR2 locus. CONCLUSIONS: This study adds seven loci to the list of genuine MS genetic risk factors and further extends the list of established loci shared across autoimmune diseases.

Comparative pharmacogenetics of multiple sclerosis: IFN-ß versus glatiramer acetate.

Various diseases require the selection of preferable treatment out of available alternatives. Multiple sclerosis (MS), an autoimmune inflammatory/neurodegenerative disease of the CNS, requires long-term medication with either specific disease-modifying therapy (DMT) - IFN-ß or glatiramer acetate (GA) - which remain the only first-line DMTs in all countries. A significant share of MS patients are resistant to treatment with one or the other DMT; therefore, the earliest choice of preferable DMT is of particular importance. A number of conventional pharmacogenetic studies performed up to the present day have identified the treatment-sensitive genetic biomarkers that might be specific for the particular drug; however, the suitable biomarkers for selection of one or another first-line DMT are remained to be found. Comparative pharmacogenetic analysis may allow the identification of the discriminative genetic biomarkers, which may be more informative for an a priori DMT choice than those found in conventional pharmacogenetic studies. The search for discriminative markers of preferable first-line DMT, which differ in carriage between IFN-ß responders and GA responders as well as between IFN-ß nonresponders and GA nonresponders, has been performed in 253 IFN-ß-treated MS patients and 285 GA-treated MS patients. A bioinformatics algorithm for identification of composite biomarkers (allelic sets) was applied on a unified set of immune-response genes, which are relevant for IFN-ß and/or GA modes of action, and identical clinical criteria of treatment response. We found the range of discriminative markers, which include polymorphic variants of CCR5, IFNAR1, TGFB1, DRB1 or CTLA4 genes, in different combinations. Every allelic set includes the CCR5 genetic variant, which probably suggests its crucial role in the modulation of the DMT response. Special attention should be given to the (CCR5*d+ IFNAR1*G) discriminative combination, which clearly points towards IFN-ß treatment choice for carriers of this combination. As a whole the comparative approach provides an option for the identification of prognostic composite biomarkers for a preferable medication among available alternatives.

Allelic combinations of immune-response genes as possible composite markers of IFN-ß efficacy in multiple sclerosis patients.

BACKGROUND: IFN-ß is widely used as the first-line disease-modifying treatment for multiple sclerosis. However, 30-50% of multiple sclerosis patients do not respond to this therapy. Identification of genetic variants and their combinations that predict responsiveness to IFN-ß could be useful for treatment prognosis. MATERIALS & METHODS: The combinations of alleles of nine polymorphic loci in immune-response genes were analyzed in 253 Russian multiple sclerosis patients as possible determinants of clinically optimal IFN-ß treatment response using APSampler software. RESULTS: Carriage of TGFB1*-509C and CCR5*d was associated with favorable IFN-ß response by itself. CCR5*d, IFNAR1*16725G, IFNG*874T and IFNB1*153T/T were the components of the combinations, associated with clinically optimal response to IFN-ß. Carriage of composite markers (CCR5*d + IFNAR1*G + IFNB1*T/T) or (CCR5*d + IFNAR1*G + IFNG*T) is beneficial for IFN-ß treatment efficacy. DISCUSSION: The data obtained provides evidence of the cumulative effect of immune-response genes on IFN-ß treatment efficacy. This joint contribution may reflect the additive effect of independent allelic variants and epistatic interactions between some of them.

Allelic combinations of immune-response genes associated with glatiramer acetate treatment response in Russian multiple sclerosis patients.

BACKGROUND: Glatiramer acetate (GA) is widely used as a first-line disease-modifying treatment for multiple sclerosis (MS). However, a significant proportion of MS patient appears to experience modest benefit from GA-treatment. Genetic variants affecting the clinical response to GA are believed to be relevant as biomarkers of GA-treatment efficiency. PATIENTS & METHODS: Nine polymorphisms in candidate genes were analyzed as possible determinants of GA response in 285 Russian MS patients. Special attention was given to identification of response-associated allelic combinations by means of the APSampler algorithm. RESULTS: No significant associations were found for individual polymorphisms. Alleles DRB1*15, TGFB1*T, CCR5*d and IFNAR1*G were the components of the combinations, of which carriage was significantly higher in nonresponders than in responders. Carriers of the most significant combinations: DRB1*15 + TGFB1*T + CCR5*d + IFNAR1*G and DRB1*15 + TGFB1*T + CCR5*d (permutation p-values: 0.0056 and 0.013, respectively) had a 14 to 15-times increased risk of ineffective response to GA therapy. DISCUSSION: The results suggest that the influence of immune-response genes on GA-induced response has a polygenic nature. The data are interpreted as evidence of additive and epistatic influences of the genes on GA efficiency for MS treatment.

Recurrent chromosomal rearrangements implicate oncogenes contributing to T-cell lymphomagenesis in Lck-MyrAkt2 transgenic mice.

The oncogene v-akt was isolated from a retrovirus that induced naturally occurring thymic lymphomas in AKR mice. We hypothesized that constitutive activation of Akt2 could serve as a first hit for the clonal expansion of malignant T-cells by promoting cell survival and genomic instability, leading to chromosome alterations. Furthermore, genes that cooperate with Akt2 to promote malignant transformation may reside at translocation/inversion junctions found in spontaneous thymic lymphomas from transgenic mice expressing constitutively active Akt2 specifically in T cells. Cytogenetic analysis revealed that thymic tumors from multiple founder lines exhibited either of two recurrent chromosomal rearrangements, inv(6)(A2B1) or t(14;15)(C2;D1). Fluorescence in situ hybridization, array CGH, and PCR analysis were used to delineate the inv(6) and t(14;15) breakpoints. Both rearrangements involved T-cell receptor loci. The inv(6) results in robust upregulation of the homeobox/transcription factor gene Dlx5 because of its relocation near the Tcrb enhancer. The t(14;15) places the Tcra enhancer in the vicinity of the Myc proto-oncogene, resulting in upregulated Myc expression. These findings suggest that activation of the Akt pathway can act as the initial hit to promote cell survival and genomic instability, whereas the acquisition of T-cell-specific overexpression of Dlx5 or Myc leads to lymphomagenesis.

Polymorphism of apolipoprotein E (APOE) and lipoprotein lipase (LPL) genes and ischaemic stroke in individuals of Yakut ethnicity.

There is evidence that most forms of ischaemic stroke (IS) result from synergistic effects of the modifiable predisposing factors and multiple genes. In the present work, we report results of case-control study of IS association with apolipoprotein E gene (APOE) (promoter and coding polymorphisms) and lipoprotein lipase gene (LPL) (presence/absence of a HindIII cutting site). We studied 107 unrelated patients of Yakut ethnicity (69 men and 38 women, mean age 58.4+/-11.5 years) with first-ever IS in carotid/middle cerebral artery regions. The control group included 101 subjects of the same ethnicity (61 men and 40 women, mean age 57.6+/-11.6 years) free of clinically detectable cerebrovascular disease, and without any history of stroke. A positive association of IS with APOE -427T allele (p=0.0012, OR=3.99) and -427T/T genotype (p=0.0005, OR=4.96) and a negative association with -427C allele (p=0.0012, OR=0.25), -427T/C genotype (p=0.0003, OR=0.18), epsilon2 allele (p=0.018, OR=0.35), epsilon2/3 genotype (p=0.017, OR=0.28) and -491A/-427C/epsilon2 haplotype (p=0.0026, OR=0.18) were observed. For atherothrombotic subgroup the same allele and genotype associations were found plus association with APOE -491A allele (p=0.026, OR=3.98). No reliable IS associations were found with LPL T+495G (HindIII) polymorphism. An association of APOE promoter polymorphisms (A-491T, T-427C) with an IS is shown in our study for the first time. Our study provides evidence for the role of APOE gene as a prognostic genetic marker for IS, especially for its atherothrombotic subtype.

Three allele combinations associated with multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an immune-mediated disease of polygenic etiology. Dissection of its genetic background is a complex problem, because of the combinatorial possibilities of gene-gene interactions. As genotyping methods improve throughput, approaches that can explore multigene interactions appropriately should lead to improved understanding of MS. METHODS: 286 unrelated patients with definite MS and 362 unrelated healthy controls of Russian descent were genotyped at polymorphic loci (including SNPs, repeat polymorphisms, and an insertion/deletion) of the DRB1, TNF, LT, TGFbeta1, CCR5 and CTLA4 genes and TNFa and TNFb microsatellites. Each allele carriership in patients and controls was compared by Fisher's exact test, and disease-associated combinations of alleles in the data set were sought using a Bayesian Markov chain Monte Carlo-based method recently developed by our group. RESULTS: We identified two previously unknown MS-associated tri-allelic combinations:-509TGFbeta1*C, DRB1*18(3), CTLA4*G and -238TNF*B1,-308TNF*A2, CTLA4*G, which perfectly separate MS cases from controls, at least in the present sample. The previously described DRB1*15(2) allele, the microsatellite TNFa9 allele and the biallelic combination CCR5Delta32, DRB1*04 were also reidentified as MS-associated. CONCLUSION: These results represent an independent validation of MS association with DRB1*15(2) and TNFa9 in Russians and are the first to find the interplay of three loci in conferring susceptibility to MS. They demonstrate the efficacy of our approach for the identification of complex-disease-associated combinations of alleles.

Metal-dependent hydrolysis of myelin basic protein by IgGs from the sera of patients with multiple sclerosis.

Homogeneous IgG fractions were obtained by chromatography of the sera of patients with multiple sclerosis (MS) on Protein G-Sepharose under conditions that remove non-specifically bound proteins. These IgGs contained several chelated metals, the relative amount of which decreases in the order: Fe>or=Ca>Cu>or=Zn>or=Mg>or=Mn>or=Pb>or=Co>or=Ni. In contrast to homogeneous IgGs of healthy individuals, Abs of MS patients effectively hydrolyzed human myelin basic protein (MBP). A minor metal-dependent fraction was obtained by chromatography of highly purified IgGs from MS patient on Chelex-100. This IgG fraction did not hydrolyze human MBP in the absence of Me(2+) ions but was activated after addition of Me(2+) ions: Mg(2+)>Mn(2+)>Cu(2+)>Ca(2+). Proteolytic activities of IgGs from other MS patients were also activated by other metal ions (Ni(2+), Fe(2+), Co(2+), Zn(2+), Pb(2+), and Co(2+)) and especially Ni(2+). Ni(2+)-activated IgGs were separated into distinct MBP-hydrolyzing fractions by chromatography on HiTraptrade mark Chelating Sepharose charged with Ni(2+). Detection of Mg(2+)-dependent proteolytic activity in the SDS-PAGE area corresponding only to IgG provided direct evidence that IgG from sera of MS patients possesses metal-dependent human MBP-hydrolyzing activity. Observed properties of MS abzymes distinguish them from other known mammalian metalloproteases and demonstrate their pronounced catalytic diversity. Metal-dependent IgGs from MS patients represent the first example of abzymes with metal-dependent proteolytic activity.

Genetic polymorphisms in human SULT1A1 and UGT1A1 genes associate with breast tumor characteristics: a case-series study.

INTRODUCTION: Estrogens are important in breast cancer development. SULT1A1 and UGT1A1 catalyze estrogen metabolism and are polymorphic. The SULT1A1*2 protein exhibits low activity, and a TA repeat within the UGT1A1 promoter alters the level of expression of the protein. We hypothesized that the SULT1A1*2 allozyme has decreased capacity to sulfate estrogens, that the SULT1A1*2 allele conferred increased capacity of cells to proliferate in response to estrogens, and that individuals with the variant SULT1A1 and UGT1A1 genotypes exhibited different breast tumor characteristics. METHODS: The capacity for SULT1A1*2 to sulfate 17beta-estradiol and the capacity for cells expressing SULT1A1*1 or SULT1A1*2 to proliferate in response to 17beta-estradiol was evaluated. A case-series study was performed in a total of 210 women with incident breast cancer, including 177 Caucasians, 25 African-Americans and eight women of other ethnic background. The SULT1A1 and UGT1A1 genotypes were determined and a logistic regression model was used to analyze genotype-phenotype associations. RESULTS: We determined that the SULT1A1*1/*1 high-activity genotype was associated with tumor size or=60 years (odds ratio = 3.70, 95% confidence interval = 1.33-10.00, P = 0.01). Individuals with both SULT1A1 and UGT1A1 high-activity genotypes had low tumor grade (odds ratio = 2.56, 95% confidence interval = 1.04-6.25, P = 0.05). Upon stratification by estrogen receptor status, significant associations were observed predominantly in estrogen receptor-negative tumors. CONCLUSION: The data suggest that genetic variation in SULT1A1 and UGT1A1 may influence breast cancer characteristics and might be important for breast cancer prognosis.

A Markov chain Monte Carlo technique for identification of combinations of allelic variants underlying complex diseases in humans.

In recent years, the number of studies focusing on the genetic basis of common disorders with a complex mode of inheritance, in which multiple genes of small effect are involved, has been steadily increasing. An improved methodology to identify the cumulative contribution of several polymorphous genes would accelerate our understanding of their importance in disease susceptibility and our ability to develop new treatments. A critical bottleneck is the inability of standard statistical approaches, developed for relatively modest predictor sets, to achieve power in the face of the enormous growth in our knowledge of genomics. The inability is due to the combinatorial complexity arising in searches for multiple interacting genes. Similar "curse of dimensionality" problems have arisen in other fields, and Bayesian statistical approaches coupled to Markov chain Monte Carlo (MCMC) techniques have led to significant improvements in understanding. We present here an algorithm, APSampler, for the exploration of potential combinations of allelic variations positively or negatively associated with a disease or with a phenotype. The algorithm relies on the rank comparison of phenotype for individuals with and without specific patterns (i.e., combinations of allelic variants) isolated in genetic backgrounds matched for the remaining significant patterns. It constructs a Markov chain to sample only potentially significant variants, minimizing the potential of large data sets to overwhelm the search. We tested APSampler on a simulated data set and on a case-control MS (multiple sclerosis) study for ethnic Russians. For the simulated data, the algorithm identified all the phenotype-associated allele combinations coded into the data and, for the MS data, it replicated the previously known findings.

Hydrolysis of myelin basic protein by IgM and IgA antibodies from the sera of patients with multiple sclerosis.

BACKGROUND: The presence of abzymes (Abzs) in human sera is a specific feature of different autoimmune pathologies. We have shown that IgGs of patients with multiple sclerosis (MS) specifically hydrolyze human myelin basic protein (hMBP). However, the presence of hMBP-hydrolyzing MS IgMs and IgAs in patients with MS has not been studied. MATERIAL/METHODS: Homogeneous IgM and IgA fractions were isolated from human sera by affinity chromatography on different adsorbents. The Ab-dependent hydrolysis of hMBP was analyzed using SDS-PAGE. RESULTS: We present evidence showing that MS IgMs and IgAs (but not Abs from the sera of healthy individuals) catalyze the hydrolysis of hMBP. Specific enzymatic activities of IgMs and sIgAs from sera of any single patient were usually significantly higher than those of IgGs. Specific inhibitors of acidic and thiol proteases demonstrated a weak effect on proteolytic activity of IgGs and IgMs. However, specific inhibitors of serine proteases (AEBSF, PMSF, and benzamidine) significantly inhibited proteolytic activity. IgMs and IgAs hydrolyze specifically both human and pig MBP but not many other tested proteins. Although the biological function of this proteolytic activity is not known, it is clear that MBP-hydrolyzing Abs may play an important role in MS pathogenesis. CONCLUSIONS: The findings display the generation by the immune systems of individual MS patients of a variety of polyclonal IgGs, IgMs, and IgAs with different proteolytic properties, which hydrolyze MBP, the major protein component of the myelin-proteolipid shell of axons and a well-known MS autoantigen.

Hydrolysis of myelin basic protein by polyclonal catalytic IgGs from the sera of patients with multiple sclerosis.

Various catalytic antibodies or abzymes have been detected recently in the sera of patients with several autoimmune pathologies, where their presence is most probably associated with autoimmunization. Recently we have shown that DNase, RNase, and polysaccharide-hydrolyzing activities are associated with IgGs from the sera of patients with multiple sclerosis (MS). Here we present evidence demonstrating that highly purified MS IgGs (but not Igs from the sera of healthy individuals) catalyze specifically hydrolysis of human myelin basic protein (hMBP). In contrast to many known proteases, IgGs do not hydrolyze many other different proteins. Specific inhibitors of acidic and thiol proteases have no remarkable effect on proteolytic activity of IgGs. However, specific inhibitor of serine (PMSF, AEBSF, and benzamidin) and metal-dependent (EDTA) proteases significantly inhibit activity of proteolytic abzymes. Interestingly, the ratio of serine-like and metal-dependent activities of MS IgGs varied very much from patient to patient. The findings speak in favor of the generation by the immune systems of individual MS patients of a variety of polyclonal anti-MBP IgGs with different catalytic properties.

A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system.

BACKGROUND: Whether for cell culture studies of protein function, construction of mouse models to enable in vivo analysis of disease epidemiology, or ultimately gene therapy of human diseases, a critical enabling step is the ability to achieve finely controlled regulation of gene expression. Previous efforts to achieve this goal have explored inducible drug regulation of gene expression, and construction of synthetic promoters based on two-hybrid paradigms, among others. RESULTS: In this report, we describe the combination of dimerizer-regulated two-hybrid and tetracycline regulatory elements in an ordered cascade, placing expression of endpoint reporters under the control of two distinct drugs. In this Dual Drug Control (DDC) system, a first plasmid expresses fusion proteins to DBD and AD, which interact only in the presence of a small molecule dimerizer; a second plasmid encodes a cassette transcriptionally responsive to the first DBD, directing expression of the Tet-OFF protein; and a third plasmid encodes a reporter gene transcriptionally responsive to binding by Tet-OFF. We evaluate the dynamic range and specificity of this system in comparison to other available systems. CONCLUSION: This study demonstrates the feasibility of combining two discrete drug-regulated expression systems in a temporally sequential cascade, without loss of dynamic range of signal induction. The efficient layering of control levels allowed by this combination of elements provides the potential for the generation of complex control circuitry that may advance ability to regulate gene expression in vivo.

Amylolytic activity of IgM and IgG antibodies from patients with multiple sclerosis.

IgG and IgM antibodies from the sera of patients with multiple sclerosis (MS) were found to possess amylolytic activity hydrolyzing alpha-(1-->4)-glucosyl linkages of maltooligosaccharides, glycogen, and several artificial substrates. Individual IgM fractions isolated from 54 analyzed patients with the clinically definite diagnoses of MS had approximately three orders of magnitude higher specific amylolytic activity than that for healthy donors, whereas IgG from only a few patients had high amylolytic activity. Strict criteria were used to prove that the amylolytic activity of IgMs and IgGs is their intrinsic property and is not due to any enzyme contamination. Fab fragments produced from IgM and IgG fractions of the MS patients displayed the same amylolytic activity. IgMs from various patients demonstrated different modes of action in hydrolyzing maltooligosaccharides.