Genetic heterogeneity in familial dysbetalipoproteinemia. The E2(lys146----gln) variant results in a dominant mode of inheritance

As determined by isoelectric focusing, most patients with familial dysbetalipoproteinemia (FD) exhibit the homozygous apolipoprotein (apo) E2E2 phenotype. Only rarely does FD develop in the more common heterozygous phenotypes E3E2 or E4E2. In fact, only 1 to 4% of the E2E2 homozygotes will develop FD. We wondered whether this reduced penetrance of FD in E2E2 homozygotes could be due to additional heterogeneity in the APOE*2 allele. In the literature a number of different mutations causing an E2 isoelectric focusing variant have been described. To study the genetic heterogeneity of the APOE gene, hybridization of enzymatically amplified genomic DNA with mutation-specific oligonucleotide probes was applied. All FD patients (n = 40) with the E2E2 phenotype appeared to be homozygous for the common E2(arg158----cys) mutation. However, all three unrelated patients with the E3E2 phenotype exhibited the rare E2(lys146----gln) mutation due to an A----C substitution at nucleotide position 3,847 of the APOE gene. This mutation was not found among normolipidemic individuals with the E2E2 (n = 13) or E3E2 phenotype (n = 120) selected from a random population sample. Family studies of the three probands heterozygous for the E*2(lys146----gln) allele showed that this rare allele predisposes to FD with high penetrance. We conclude that FD is a genetically heterogeneous disease entity, displaying a recessive mode of inheritance with strongly reduced penetrance in case of the common E2(arg158----cys) variant and with a dominant mode of inheritance with high penetrance in case of the rare E2(lys146----gln) mutant. It should be noted that in this dominant form presymptomatic diagnosis is possible.     

Apolipoprotein E3-Leiden contains a seven-amino acid insertion that is a tandem repeat of residues 121-127

Apolipoprotein (apo) E3-Leiden is a variant of apoE that is associated with dominant expression of type III hyperlipoproteinemia and that is defective in binding to the low density lipoprotein receptor. Therefore, the structure of apoE3-Leiden was investigated. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis apoE3-Leiden and its 22-kDa amino-terminal thrombolytic fragment migrated with a higher than normal apparent molecular weight. The structural abnormality of apoE3-Leiden was determined by sequencing its CNBr-, tryptic-, and Staphylococcus aureus V8 protease-generated peptides. In contrast to normal apoE3, which has a cysteine at residue 112, apoE3-Leiden does not contain any cysteine and has an arginine at position 112 (as does apoE4, which also completely lacks cysteine). The basis for the molecular weight difference was determined to be a seven-amino acid insertion that is a tandem repeat of residues 121-127 of normal apoE3, i.e. Glu-Val-Gln-Ala-Met-Leu-Gly, resulting in apoE3-Leiden having 306 amino acids rather than 299. The negatively charged glutamyl residues within the insertion compensates for the arginine substitution at residue 112; thus apoE3-Leiden focuses in the E3 position. The low density lipoprotein receptor binding activities of both intact apoE3-Leiden and its 22-kDa thrombolytic fragment were determined in an in vitro assay. Although apoE3-Leiden had only about 25% of normal binding activity, its 22-kDa thrombolytic fragment had nearly normal binding, suggesting that the carboxyl-terminal domain of apoE3-Leiden modulates the receptor binding function of its amino-terminal domain.     

Apolipoprotein E3-Leiden. A new variant of human apolipoprotein E associated with familial type III hyperlipoproteinemia

Summary A variant of apolipoprotein E, denoted apo E3-Leiden, has been identified in a 41-year-old male suffering from type III hyperlipoproteinemia with xanthomatosis. Apo E3-Leiden focus in the E3 position. In contrast with normal apo E3, apo E3-Leiden is defective in binding to the low density lipoprotein (LDL) receptor and does not contain cysteine as evaluated by cysteamine treatment of very low density lipoprotein followed by isoelectric focusing and conventional protein staining and by amino acid analysis. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, apo E3-Leiden displays an electrophoretic mobility intermediate to that of normal apo E3 and apo E2 (Arg₁₅₈Cys). The mother and four siblings of the proband also have apo E3-Leiden and hyperlipoproteinemia type III; three of them with xanthomatosis. Two siblings do not show apo E3-Leiden in their VLDL fraction and do not have hyperlipoproteinemia type III. In the VLDL fractions of all affected family members only the presence of apo E3-Leiden could be detected after cysteamine treatment and isoelectric focusing followed by conventional protein staining. However, isoelectric focusing of cysteaminetreated sera followed by immunoblotting, using anti-apo E antiserum as first antiserum, demonstrates the presence of low amounts of normal apo E3 in addition to apo E3-Leiden in serum of the affected family members. These results indicate that all affected family members are heterozygotes E3/E3-Leiden and suggest that in this family type III hyperlipoproteinemia is transmitted as a dominant trait.     

Chylomicron remnant uptake in the livers of mice expressing human apolipoproteins E3, E2 (Arg158-->Cys), and E3-Leiden

Apolipoprotein E2 (apoE2) and apoE3-Leiden cause chylomicron remnant accumulation (type III hyperlipidemia). However, the degree of dyslipidemia and its penetrance are different in humans and mice. Remnant uptake by isolated liver from apoE-/- mice transgenic for human apoE2, apoE3-Leiden, or apoE3 was measured. In the presence of both LDL receptor (LDLR) and LDL receptor-related protein (LRP), remnant uptake was apoE3>E3-Leiden>E2 mice. Absence of LDLR reduced uptake in apoE3 and apoE3-Leiden-secreting livers but not in apoE2-secreting livers. LRP inhibition with receptor-associated protein reduced uptake in apoE3- and apoE2-secreting livers, but not in apoE3-Leiden-secreting livers, regardless of the presence of LDLR. Fluorescently labeled remnants clustered with LRP in apoE3-secreting livers only in the absence of LDLR, but clustered in livers that expressed apoE2 even in the presence of LDLR, and did not cluster with LRP in livers of apoE3-Leiden even in the absence of LDLR. Remnants were reconstituted with the three human apoE isoforms. Removal by liver of mApoe-/-/mldlr-/- mice expressing the human LDLR was slightly greater than removal in the previous experiments with apoE3>E2> E3-Leiden. Thus, in vivo, human apoE2 is cleared primarily by LRP, apoE3-Leiden is cleared only by the LDLR, and apoE3 is cleared by both.     

Mutation spectra of TA*, the major photoproduct of thymidylyl-(3'5')-deoxyadenosine, in Escherichia coli under SOS conditions.

The biological activity of TA*, the major photoproduct of thymidylyl-(3',5')-deoxyadenosine, has remained speculative since it was identified a decade ago. To determine the mutagenicity of TA* in Escherichia coli, we constructed the replicative form of an M13mp18-derived phage containing TA* in the (-)-strand by polymerase-catalyzed elongation of a TA*-containing 49mer opposite a uracil-containing (+)-strand of the phage. The in vitro synthesis mixture was transfected into an ung+, phr- E.coli host and the progeny were screened with a hybridization probe unique for the (-)-strand. TA* was found to block DNA replication substantially in the absence of SOS, but under SOS, TA* was bypassed more efficiently and was highly mutagenic. Among 56 analyzed (-)-strand progeny from two transfections, 46 (82%) were mutants, including six (11%) tandem mutants. The most abundant mutation was a 3'A-->T substitution (31/46, 56%). The possible biological consequences of TA* formation in the highly conserved TATA box consensus sequence on gene expression are discussed in light of the mutagenicity of TA*.     

Fenofibrate increases HDL-cholesterol by reducing cholesteryl ester transfer protein expression

In addition to efficiently decreasing VLDL-triglycerides (TGs), fenofibrate increases HDL-cholesterol levels in humans. We investigated whether the fenofibrate-induced increase in HDL-cholesterol is dependent on the expression of the cholesteryl ester transfer protein (CETP). To this end, APOE*3-Leiden (E3L) transgenic mice without and with the human CETP transgene, under the control of its natural regulatory flanking regions, were fed a Western-type diet with or without fenofibrate. Fenofibrate (0.04% in the diet) decreased plasma TG in E3L and E3L.CETP mice (-59% and -60%; P < 0.001), caused by a strong reduction in VLDL. Whereas fenofibrate did not affect HDL-cholesterol in E3L mice, fenofibrate dose-dependently increased HDL-cholesterol in E3L.CETP mice (up to +91%). Fenofibrate did not affect the turnover of HDL-cholesteryl ester (CE), indicating that fenofibrate causes a higher steady-state HDL-cholesterol level without altering the HDL-cholesterol flux through plasma. Analysis of the hepatic gene expression profile showed that fenofibrate did not differentially affect the main players in HDL metabolism in E3L.CETP mice compared with E3L mice. However, in E3L.CETP mice, fenofibrate reduced hepatic CETP mRNA (-72%; P < 0.01) as well as the CE transfer activity in plasma (-73%; P < 0.01). We conclude that fenofibrate increases HDL-cholesterol by reducing the CETP-dependent transfer of cholesterol from HDL to (V)LDL, as related to lower hepatic CETP expression and a reduced plasma (V)LDL pool.     

Apolipoprotein E polymorphism in India: high APOE*E3 allele frequency in Ramgarhia of Punjab

High resolution two dimensional gel electrophoresis with the combination of isoelectric focusing (IEF) and density gradient sodium dodecyl-polyacrylamide gel electrophoresis (DG-PAGE) have been employed to investigate the distribution of APOE in Ramgarhia (n = 80) and Ramdasia (n = 70) of Punjab, India. Three alleles APOE*E2, APOE*E3 and APOE*E4 were observed in Ramgarhia and Ramdasia with the frequencies of 0.031, 0.913, 0.056 and 0.043, 0.886 and 0.071, respectively. Higher heterozygosity (20.8%) in Ramdasia reflects greater variation at the APOE locus. The APOE*E3 allele is found to be the highest (0.913) in Ramgarhia in comparison to forty-one populations of the world. A decreasing cline from south to north was evident for *E2 and *E4 allele frequencies (y = -0.002x + 0.141, r = 0.78 and y = -0.004x + 0.229, r = 0.83, respectively, and an increasing cline for the *E3 allele towards north was observed (y = 0.006x + 0.629, r = 0.82) in Asia.     

CETP expression reverses the reconstituted HDL-induced increase in VLDL

Human data suggest that reconstituted HDL (rHDL) infusion can induce atherosclerosis regression. Studies in mice indicated that rHDL infusion adversely affects VLDL levels, but this effect is less apparent in humans. This discrepancy may be explained by the fact that humans, in contrast to mice, express cholesteryl ester transfer protein (CETP). The aim of this study was to investigate the role of CETP in the effects of rHDL on VLDL metabolism by using APOE*3-Leiden (E3L) mice, a well-established model for human-like lipoprotein metabolism. At 1 h after injection, rHDL increased plasma VLDL-C and TG in E3L mice, but not in E3L mice cross-bred onto a human CETP background (E3L.CETP mice). This initial raise in VLDL, caused by competition between rHDL and VLDL for LPL-mediated TG hydrolysis, was thus prevented by CETP. At 24 h after injection, rHDL caused a second increase in VLDL-C and TG in E3L mice, whereas rHDL had even decreased VLDL in E3L.CETP mice. This secondary raise in VLDL was due to increased hepatic VLDL-TG production. Collectively, we conclude that CETP protects against the rHDL-induced increase in VLDL. We anticipate that studies evaluating the anti-atherosclerotic efficacy of rHDL in mice that are naturally deficient for CETP should be interpreted with caution, and that treatment of atherogenic dyslipidemia by rHDL should not be combined with agents that aggressively reduce CETP activity.     

Histidine 190-D1 and glutamate 189-D1 provide structural stabilization in photosystem II

In photosynthesis, photosystem II (PSII) conducts the light-driven oxidation of water to oxygen. Tyrosine Z is Tyr 161 of the D1 polypeptide; Z acts as an intermediary electron carrier in water oxidation. In this report, EPR spectroscopy was used to study the effect of His 190 and Glu 189 on Z* yield and reduction kinetics. Neither mutation has a significant impact on the EPR line shape of Z*. At room temperature and pH 7.5, the E189Q-D1 mutation has a single turnover Z* yield that is 84% compared to wild-type. The H190Q-D1 mutation decreases the Z* yield at room temperature by a factor of 2.6 but has a more modest effect (factor of 1.6) at -10 degrees C. The temperature dependence is shown to be primarily reversible. Neither mutation has a dramatic effect on Z* decay kinetics. The Z* minus Z FT-IR spectrum, recorded at pH 7.5 on H190Q, reveals perturbations, including an increased spectral contribution from a PSII chlorophyll. The Z* minus Z FT-IR spectrum, recorded at pH 7.5 on E189Q, shows perturbations, including a decreased contribution from the carboxylate side chain of a glutamate or aspartate. Temperature-dependent changes in H190Q-D1 and E189Q-D1 Z. yield are attributed to a reversible conformational change, which alters the electron-transfer rate from Z to P(680)(+). On the basis of these results, we conclude that H190 and E189 play a role in the structural stabilization of PSII. We postulate that some or all of the phenotypic changes observed in H190Q and E189Q mutants may be caused by structural alterations in PSII.     

Thymus-dependent network responses to a monoclonal cross-reactive antiidiotypic antibody.

BALB/c mice were inoculated i.p. with a cross-reactive anti-Idiotypic mAb (designated FD5-1) in the absence of Ag or adjuvants. Injection with unmodified FD5-1 resulted in the induction of serum antibodies reactive with both FD5-1 (Ab3) and the hapten DNP (Ab1'). Endpoint titers of the Ab3 response showed an increase in serum IgM, which was dose-responsive to both the number of injections and the amount of FD5-1 antibody injected. The serum IgM Ab3 response was found to be thymus dependent and idiotypically specific for FD5-1. Athymic mice injected with FD5-1 were unable to produce a serum IgM Ab3 response, whereas their euthymic littermates produced strong Ab3 responses. Serum Ab3 responses and Ab1' were detectable only in the IgM isotype; no specific IgG responses were observed. Indeed, IgG recognized by FD5-1 appeared to be suppressed by FD5-1. Injection of mice with FD5-1 modulated serum IgM responses to DNP, (4-hydroxy-3-nitrophenyl)acetyl (NP), 4-ethoxymethylene-2-phenyloxazol-5-one (OX), phosphorylcholine (PC), and alpha 1,3-dextran (DEX) in a thymus-dependent manner. FD5-1 injection induced IgM responses against DNP, (4-hydroxy-3-nitrophenyl)acetyl (NP), 4-ethoxymethylene-2-phenyloxazol-5-one, and DEX but decreased IgM binding to PC. No detectable Ab1' responses to any of the aforementioned molecules were found when the same sera were probed for IgG. The specificity of serum Ab1' from FD5-1-injected mice was evaluated by antigenic inhibition. Binding of serum Ab1' to DNP-BSA was inhibitable by DNP-lysine, whereas equivalent concentrations of lysine alone had no inhibitory effect. The antigenic specificity of IgM from normal serum binding to PC-BSA was demonstrated by inhibition with free PC, and the binding of Ab1' from FD5-1-injected mice to DEX-coated plates was shown to be inhibitable by DEX. We have described in vivo network perturbation in adult BALB/c mice injected with anti-Id antibody in the absence of Ag or adjuvants. Our findings show that injection of the cross-reactive anti-Id FD5-1 can induce thymus-dependent Ag-specific responses. In two systems where FD5-1 functions as an anti-anti-anti-Id antibody (PC and DEX), thymus-dependent responses were also observed. FD5-1 injection suppressed antibodies binding to PC, whereas DEX-specific responses were induced.     

Association between the APOE*4 allele and atherosclerosis is age dependent among Argentine males

Several studies have shown evidence of an association between the *4 allele of apolipoprotein E (APOE) and coronary heart disease (CHD) in different populations. We determined the APOE genotype and total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDLC) values in 189 patients with angiographically evaluated atherosclerosis. The APOE*4 allele was found to be statistically significantly more frequent (odds ratio, 1.93; 95% confidence interval, 1.12-3.32) among male patients than in a randomly chosen population-based sample. No significant difference was found when female patients were compared to the general population. The APOE*4 allele was found primarily among young (30-45-year-old) male patients (p < 0.04). Despite the ascending linear tendency of the mean TC values for genotypes APOE*2/*3, APOE*3/*3, and APOE*3/ *4 reported in our case population, no differences were observed among our patients. We conclude that the APOE*4 allele is associated with an increased risk for atherosclerotic vascular disease, that this association has an age-dependent effect, and that it acts as a genetic factor that increases susceptibility to developing the disease in young to middle-aged male adults in our population.     

Screening for familial dysautonomia in Israel: evidence for higher carrier rate among Polish Ashkenazi Jews

Familial dysautonomia (FD) is an autosomal recessive disorder characterized by hereditary sensory and autonomic neuropathies. Although extremely rare in most populations, FD is common among Ashkenazi Jews (AJ), with a calculated carrier frequency of 1 in 30, based on disease prevalence. The gene for FD was recently identified as IKBKAP. One major mutation (IVS2 + 6T --> C) is responsible in >99.5% of cases among AJ. The purpose of this study was to determine the actual frequency of FD carriers in the AJ population in Israel and to determine whether carriers are more frequent among a subpopulation of AJ from Poland. The study group included 1267 Jews of Ashkenazi origin who were referred for routine DNA screening tests. These included 1100 individuals who were full AJ and 167 who were part AJ. None had a family history of FD. Mutation analysis for (IVS2 + 6T --> C) was performed by PCR amplification followed by restriction enzyme analysis. All positive cases were confirmed by DHPLC WAVE( trade mark ). Among the 1100 full AJ tested, 34 were found to be FD carriers (1:32). The incidence of mutation carriers was significantly higher in AJ of Polish descent (1:18) compared to AJ of non-Polish descent (1:99). Among the 167 individuals who were part AJ, there were 3 carriers (1:56). The incidence of FD among AJ, particularly those of Polish background, warrants population screening. Population screening may be performed by denaturing high-performance liquid chromatography.     

Hybridization specificity, enzymatic activity and biological (Ha-ras) activity of oligonucleotides containing 2,4-dideoxy-beta-D-erythro-hexopyranosyl nucleosides.

Antisense oligonucleotides with a 2,4-dideoxyhexopyranosyl nucleoside incorporated at the 3'-end and at a mutation site of the Ha-ras oncogene mRNA were synthesized. Melting temperature studies revealed that an A*-G mismatch is more stable than an A*-T mismatch with these hexopyranosyl nucleosides incorporated at the mutation site. The oligonucleotides are stable against enzymatic degradation. RNase H mediated cleavage studies revealed selective cleavage of mutated Ha-ras mRNA. The oligonucleotide containing two pyranose nucleosides at the penultimate position activates RNase H more strongly than natural oligonucleotides. No correlation, however, was found between DNA - DNA or RNA - DNA melting temperatures and RNase H mediated cleavage capacity. Although the A*-G mismatch gives more stable hybridization than the A*-T base pairing, only the oligonucleotides containing an A*-T base pair are recognized by RNase H. This modification is situated 3 base pairs upstream to the cleavage site. Finally, the double pyranose modified oligonucleotide was able to reduce the growth of T24 cells (bladder carcinoma) while the unmodified antisense oligonucleotide was not.     

Melanocortin-1 receptor variant R151C modifies melanoma risk in Dutch families with melanoma

Germline mutations of the cell-cycle regulator p16 (also called "CDKN2A") in kindreds with melanoma implicate this gene in susceptibility to malignant melanoma. Most families with familial atypical multiple-mole melanoma (FAMMM) who are registered at the Leiden dermatology clinic share the same p16-inactivating deletion (p16-Leiden). Incomplete penetrance and variable clinical expression suggest risk modification by other genetic and/or environmental factors. Variants of the melanocortin-1 receptor (MC1R) gene have been shown to be associated with red hair, fair skin, and melanoma in humans. Carriers of the p16-Leiden deletion in Dutch families with FAMMM show an increased risk of melanoma when they also carry MC1R variant alleles. The R151C variant is overrepresented in patients with melanoma who are from families with the p16-Leiden mutation. Although some of the effect of the R151C variant on melanoma risk may be attributable to its effect on skin type, our analyses indicate that the R151C variant contributes an increased melanoma risk even after statistical correction for its effect on skin type. These findings suggest that the R151C variant may be involved in melanoma tumorigenesis in a dual manner, both as a determinant of fair skin and as a component in an independent additional pathway.     

Occurrence of a 2-bp (AT) deletion allele and a nonsense (G-to-T) mutant allele at the E2 (DBT) locus of six patients with maple syrup urine disease: multiple-exon skipping as a secondary effect of the mutations.

We have identified two novel mutant alleles in the transacylase (E2) gene of the human branched-chain alpha-keto acid dehydrogenase (BCKAD) complex in 6 of 38 patients with maple syrup urine disease (MSUD). One mutation, a 2-bp (AT) deletion in exon 2 of the E2 gene, causes a frameshift downstream of residue (-26) in the mitochondrial targeting presequence. The second mutation, a G-to-T transversion in exon 6 of the E2 gene, produces a premature stop codon at Glu-163 (E163*). Transfection of constructs harboring the E163* mutation into an E2-deficient MSUD cell line produced a truncated E2 subunit. However, this mutant E2 chain is unable to assemble into a 24-mer cubic structure and is degraded in the cell. The 2-bp (AT) deletion and the E163* mutant alleles occur in either the homozygous or compound-heterozygous state in the 6 of 38 unrelated MSUD patients studied. Moreover, an array of precise single- and multiple-exon deletions were observed in many amplified E2 mutant cDNAs. The latter results appear to represent secondary effects on RNA processing that are associated with the MSUD mutations at the E2 locus.     

A new recessive ametapodia mutation in the chicken (Gallus domesticus)

An apparently new mutation that is associated with abnormal limb development appeared in a strain of Light Brown Leghorn chickens. Mutants are characterized by the complete absence of the tarsometatarsals, while severely hypoplastic development of the metacarpals is also present. The phenotype of the new mutant (ametapodia-2) closely resembles ametapodia-1, described in 1967, but ametapodia-2 is inherited as an autosomal recessive (AMET*A), while ametapodia-1 was associated with an incompletely dominant gene (MP*A). Only heterozygous ametapodia-1 (MP*N/MP*A) were viable and able to reproduce, while homozygous ametapodia-2 mutants do not normally survive beyond 2-4 days of age. The shankless mutation (SHL*S) also reduces development of the metatarsal and metacarpal bones and has been shown to be associated with a pericentric inversion of chromosome 2. No obvious cytologic abnormality was apparent in ametapodia-2 birds, and offspring of a cross between AMET*A carriers and shankless birds were normal, indicating that the two mutations are not alleles. Ametapodia-1 (MP*A) was found to be linked to the rose comb locus (R) by 16 crossover units. Linkage test matings between AMET*A and (R*R) showed independent segregation, strongly suggesting that the mutation occurred at a relatively distant locus and therefore is probably not allelic to MP*A.     

Glucose-induced conformational changes in glucokinase mediate allosteric regulation: transient kinetic analysis

The transient kinetics of glucose binding to glucokinase (GK) was studied using stopped-flow fluorescence spectrophotometry to investigate the underlying mechanism of positive cooperativity of monomeric GK with glucose. Glucose binding to GK was shown to display biphasic kinetics that fit best to a reversible two-step mechanism. GK initially binds glucose to form a transient intermediate, namely, E* x glucose, followed by a conformational change to a catalytically competent E x glucose complex. The microscopic rate constants for each step were determined as follows: on rate k1 of 557 M(-1) s(-1) and off rate k(-1) of 8.1 s(-1) for E* x glucose formation, and forward rate k2 of 0.45 s(-1) and reverse rate k(-2) of 0.28 s(-1) for the conformational change from E* x glucose to E x glucose. These results suggest that the enzyme conformational change induced by glucose binding is a reversible, slow event that occurs outside the catalytic cycle (kcat = 38 s(-1)). This slow transition between the two enzyme conformations modulated by glucose likely forms the kinetic foundation for the allosteric regulation. Furthermore, the kinetics of the enzyme conformational change was altered in favor of E x glucose formation in D2O, accompanied by a decrease in cooperativity with glucose (Hill slope of 1.3 in D2O vs 1.7 in H2O). The deuterium solvent isotope effects confirm the role of the conformational change in the magnitude of glucose cooperativity. Similar studies were conducted with GK activating mutation Y214C at the allosteric activator site that is likely involved in the protein domain rearrangement associated with glucose binding. The mutation enhanced equilibrium glucose binding by a combination of effects on both the formation of E* x glucose and an enzyme conformational change to E x glucose. Kinetic simulation by KINSIM supports the conclusion that the kinetic cooperativity of GK arises from slow glucose-induced conformational changes in GK.     

Conformation of D-Orn2-containing cycloanalogs of enkephalins in dimethylsulfoxide solution

The sterically acceptable structures of cyclo(2 delta----5)[D-Orn2, Pro5]- and cyclo(2 delta----5)[D-Orn2, Leu5]enkephalin (CE1 and CE2) consistent with NMR data including coupling constants, temperature dependencies of chemical shifts for amide protons and NOE values have been found by use of energy calculations in terms of rigid valence geometry and refined by the MM2 procedure. It has been shown that the major trans-isomer (with respect to Phe4-Pro5 bond) of CE1 in solution corresponds only to the FD*F*AA type of peptide backbone, and the minor cis-isomer of CE1 corresponds only to the FE*D*DF type. The less conformationally rigid CE2 analogue apparently exists in solution in the dynamic conformational equilibrium with preference of FD*C*AA type of the backbone structure. The obtained data on CE1 and CE2 space structures have been used for interpretating results of their biological testing.     

SSCP analysis at the bovine CSN3 locus discriminates six alleles corresponding to known protein variants (A, B, C, E, F, G) and three new DNA polymorphisms (H, I, A1)

A high resolution SSCP protocol was developed for simultaneous discrimination of the known CSN3 alleles A, B, C, E, F and G. Furthermore, three new DNA polymorphisms were identified in different Bos taurus and Bos indicus breeds or crosses. Mendelian segregation was shown for two of these polymorphisms (named CSN3*H and 1), and the third (named CSN3*A1) was found in unrelated animals, thus indicating the presence of three additional alleles at the bovine CSN3 locus. DNA sequencing revealed single mutations that led to a Thr/Ile substitution in amino acid position 135 for CSN3*H and to a Ser/Ala substitution in position 104 of the deduced amino acid sequence of CSN3*1 (GenBank accession numbers AF105260 and AF121023) compared to CSN3*A. In CSN3*A1, a silent mutation in the third codon position of Pro150 was found (GenBank accession number AF092513).