Comment on "Observation of oscillatory magnetic order in the antiferromagnetic superconductor HoNi2Ba2C"
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Journal Article↗
Biomedical subjects
A Goldman
Publications and source records attributed to A Goldman.
Diffuse scattering and phason elasticity in the AlPdMn icosahedral phase.
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Journal Article↗
New crystal forms of Escherichia coli and Saccharomyces cerevisiae soluble inorganic pyrophosphatases.
We have obtained new crystal forms of Escherichia coli and Saccharomyces cerevisiae soluble inorganic pyrophosphatase with and without substrate, competitive inhibitor and divalent cation. They diffract to higher resolution than any forms previously reported. The best E. coli crystals are in space group R32 with cell dimensions of 111.4 x 111.4 x 76.6 A and diffract to 2.0 A. The best S. cerevisiae crystals were grown from a mixture of PEG 1000 and 4000 in the presence of metal ions. They are in space group P2(1)2(1)2(1), have cell dimensions of 54.2 x 68.5 x 161.7 A and diffract to 1.8 A.
Journal Article↗
Cold lability of the mutant forms of Escherichia coli inorganic pyrophosphatase.
The variants of Escherichia coli pyrophosphatase carrying the substitutions Glu20-->Asp, His136-->Gln or His140-->Gln are inactivated, in contrast to the wild-type enzyme, at temperatures below 25 degrees C: their activity measured at 25 degrees C decreases with decreasing the temperature of the stock enzyme solution. The inactivation is completely reversible and is explained by cold-induced dissociation of these hexameric enzymes into less active trimers.
Aspartic Acid↗
Structure and function analysis of Escherichia coli inorganic pyrophosphatase: is a hydroxide ion the key to catalysis?
Using site-directed mutagenesis, we have completed replacing all 17 putative active site residues of Escherichia coli inorganic pyrophosphatase (PPase). We report here the production of 11 new variant proteins and their initial characterization, including thermostability, hydrophobicity, oligomeric structure, and specific activity at pH 8. Studies of the pH-rate profiles of 12 variants containing substitutions for potentially essential residues showed that the effect of the mutation was always to increase the pKa of a basic group essential for both substrate binding and catalysis by 1-3 pH units. The D70E variant had the lowest activity at all pHs; the K29R, R43K, and K142R variants also had low kcat/Km values. The principal effect seen in the other variant proteins was higher and sharper pH optima; their pH-independent kcat and kcat/Km values changed at most by a factor of 8. Our results suggest that the most likely candidate for the essential basic group affected by all mutations in the active site is a hydroxide ion stabilized by coordination to the essential Mg2+ ions. Analyzing our results using the structure recently obtained for E. coli PPase [Kankare et al. (1994) Protein Eng. 7, 823-830] led us to identify a group of residues, centered around Asp70 and including Tyr55, Asp65, Asp67, Asp102, and Lys104, that we believe binds the magnesium ions that are critical for the activity, possibly by stabilizing the essential hydroxide. Others, including Lys29, Arg43, and Lys142, are more spread out and more positively charged. They appear to be involved in binding substrate and product. Tyr55 is also a key part of the hydrophobic core of E. coli PPase; when it or residues that interact with it are conservatively mutated, there are changes in the overall structure of the enzyme as assayed by thermostability, hydrophobicity, or oligomeric structure.
Bacterial Proteins↗
Effect of D97E substitution on the kinetic and thermodynamic properties of Escherichia coli inorganic pyrophosphatase.
Aspartic acid 97 in the inorganic pyrophosphatase of Escherichia coli (E-PPase) has been identified as an evolutionarily conserved residue forming part of the active site [Cooperman et al. (1992) Trends Biochem. Sci. 17, 262-266]. Here we determine the effect of D97E substitution on several kinetic and thermodynamic properties of E-PPase, including rate and equilibrium constants for enzyme-catalyzed PPi.Pi equilibration at pH 7.2 and 8.0, Mg2+ affinity in the presence and absence of substrate, and the Mg2+ and pH dependence of kcat and Km. We find the major effects of D97E substitution are to (a) decrease markedly the pH-independence rates of both PPi hydrolysis and, especially, PPi resynthesis on the enzyme, (b) selectively destabilize both the EMg4PPi complex and the transition state between this complex and the EMg2(MgPi)2 complex, (c) raise the pKa of the basic group "essential" for PPi hydrolysis and for productive PPi binding by 1.5 and > 2.2 log units, respectively, (d) distort a site to which Mg2+ binds in the absence of substrate such that occupancy of the site by Mg2+ no longer confers enzymatic activity, and (e) decrease the affinity of one of the two Mg2+ ions that binds to enzyme in the presence of substrate. That this multiplicity of effects arises from a single Asp to Glu substitution suggests, in the absence of any evidence for a generalized structural change, a tightly integrated active site in which the perturbation induced by conservative substitution at a single location can have widespread functional effects.
Bacterial Proteins↗
Long-lasting functional unresponsiveness induced by a milk-transmitted Mls-1a-like superantigen.
We have recently shown (Piazzon et al. (1994) J. Immunol. 153, 1553) that foster-nursing of BALB/c mice on F1 Mls-1bxa mothers induce the progressive deletion of V beta 6+ and 8.1+ T cells in 50% of the mice. Preceding clonal deletion, a state of functional inactivation of CD4+ T cells to Mls-1a and anti-V beta 6 antibodies was detected in young mice. In the present paper we show that foster-nursing of BALB/c mice on (BALB/cxAKR)FI mothers is able to induce alterations in T cell reactivity in the non-deletor mice. Lymph node cells from foster-nursed mice show a decreased proliferative level against anti-V beta 6 antibodies and a diminished response in MLR and in CTL assays. The proliferative responses to either OVA or Con-A are also reduced. This state of functional inactivation is detected even in 6-month-old foster-nursed mice. Thus, the transmission through milk of the Mls-1a-like superantigen correlates in the non-deletor mice with a long-lasting state of functional inactivation and a decreased immune reactivity.
Animals↗
Insights from monitoring the CPCRA didanosine/zalcitabine trial. Terry Beirn Community Programs for Clinical Research on AIDS.
The design, conduct, and analysis of clinical trials that evaluate the safety and efficacy of treatment interventions in patients with HIV infection provide many scientific challenges. A recently completed randomized trial of didanosine (ddI) and zalcitabine (ddC), sponsored by the Terry Beirn Community Programs for Clinical Research on AIDS (CPCRA), is an especially valuable resource for illustrating these challenging issues and for providing insights into how they might be properly addressed. Establishing equivalence of treatment effects on clinical efficacy end points is illustrated through the use of the confidence interval approach. The striking changes in treatment efficacy results that occurred during the course of the CPCRA trial provide important insights into how a data and safety monitoring board can reduce the risk of inappropriate early study termination. The trial also provides valuable insights into how treatment effects should be assessed, revealing inconsistencies between effects on the CD4 surrogate end point and effects on primary clinical efficacy end points and showing the incompleteness of the standardly employed definition of AIDS progression. Finally, the results of this ddI/ddC trial are used to examine the role of covariate adjustment.
Antiviral Agents↗
A maternally-inherited alteration in the T cell repertoire of BALB/c mice.
A number of milk-borne exogenous mammary tumor viruses (MMTV) infect mice shortly after birth and, when expressed, produce superantigens. The expression of these superantigens mediate the progressive deletion of T cells expressing specific V beta products. Here we describe a maternally-inherited alteration in the T cell repertoire in one colony of BALB/c mice which has not been reported up to now. This alteration involves the deletion of V beta 2+ and 14+ CD4+ T cells and correlates with a high incidence of mammary tumors, suggesting the involvement of a superantigen(s) probably transmitted through an exogenous MMTV in milk.
Animals↗
New founder haplotypes at the myotonic dystrophy locus in southern Africa.
The association between normal alleles at the CTG repeat and two nearby polymorphisms in the myotonin protein kinase gene, the Alu insertion/deletion polymorphism and the myotonic dystrophy kinase (DMK)(G/T) intron 9/HinfI polymorphism, has been analyzed in South African Negroids, a population in which myotonic dystrophy (DM) has not been described. South African Negroids have a CTG allelic distribution that is significantly different from that in Caucasoids and Japanese: the CTG repeat lengths of > or = 19 are very rare. The striking linkage disequilibrium between specific alleles at the Alu polymorphism (Alu(ins) and Alu(del)), the HinfI polymorphism (HinfI-1 and HinfI-2), and the CTG repeat polymorphism seen in Caucasoid (Europeans and Canadians) populations was also found in the South African Negroid population. Numerous haplotypes, not previously described in Europeans, were, however, found. It thus seems likely that only a small number of these "African" chromosomes were present in the progenitors of all non-African peoples. These data provide support for the "out of Africa" model for the origin of modern humans and suggest that the rare ancestral DM mutation event may have occurred after the migration from Africa, hence the absence of DM in sub-Saharan Negroid peoples.
Africa, Southern↗
Molecular analysis of the CTG trinucleotide repeat in South African myotonic dystrophy families--implications for diagnosis and counselling.
Myotonic dystrophy is associated with an increased number of CTG repeats in the 3' untranslated region of the myotonin protein kinase gene. The recent elucidation of the molecular basis of myotonic dystrophy has, for the first time, made a specific molecular diagnosis of this condition a possibility. Ten South African families were analysed at the molecular level, using both PCR and Southern blot analyses for the detection of the trinucleotide repeat. Expansion of this repeat was found in 9 families and in 2 cases the grandparental origin, which was previously unknown, could be determined. It is now possible to counsel these families more effectively and to identify individuals, particularly women, who are at risk of passing on the DM mutation.
Blotting, Southern↗
Transmission of an Mls-1a-like superantigen to BALB/c mice by foster-nursing on F1 Mls-1bxa mothers. Sex-influenced onset of clonal deletion.
Foster nursing of BALB/c (Mls-1b) mice on (BALB/cxAKR/J)F1 and (BALB/cxDBA/2)F1 (Mls-1bxa), but not on (BALB/cxC57Bl/6)F1 or (BALB/cxC3H/He)F1 (Mls-1bxb mothers, induced the progressive deletion of V beta 6+ and V beta 8.1+ T cells in 50% of the litter. The onset of this Mls-1a-like clonal deletion was markedly sex-influenced, being earlier in females (8-10 wk of age) than in males (32 wk). In both sexes, CD4+ V beta 6+ cells were more affected than CD8+ V beta 6+ cells. Decreases in the percentage of V beta 6+ cells were detected simultaneously in the thymus, lymph nodes, and peripheral blood. Preceding clonal deletion, functional unresponsiveness of CD4+ T cells to Mls-1 a Ags and to anti-V beta 6 Abs could be detected in most young male and female mice. The transmission of the Mls-1a-like superantigen through foster-nursing on (BALB/cxAKR/J)F1 mice correlated with the presence in milk of the mouse mammary tumor virus envelope protein gp52.
Animals↗
Crystal structure of chloromuconate cycloisomerase from Alcaligenes eutrophus JMP134 (pJP4) at 3 A resolution.
Chloromuconate cycloisomerase (E.C. 5.5.1.7) is an enzyme involved in the 2,4-dichlorophenoxyacetate degradation pathway of Alcaligenes eutrophus JMP134 (pJP4). The crystal structure of this protein was determined at 3 A resolution by molecular-replacement techniques using atomic coordinates from the reported crystal structure of the homologous muconate cycloisomerase (E.C. 5.5.1.1) from Pseudomonas putida as the search model (42% identical positions in the sequences). Structure refinement by simulated-annealing and restrained least-squares techniques converged at R = 0.195. In the crystals studied, space group I4, the protein is present as two octamers per unit cell with two subunits per asymmetric unit. Each subunit consists of two globular domains, one of which forms an alpha/beta-barrel. Comparison of this structure with that of muconate cycloisomerase reveals the reasons for the altered substrate specificity of chloromuconate cycloisomerase. Marked differences are observed in polarity, accessibility and hydrogen-bonding potential in the channel leading into the active site as well as in the active center itself.
Journal Article↗
The structure of E.coli soluble inorganic pyrophosphatase at 2.7 A resolution.
The structure of E.coli soluble inorganic pyrophosphatase has been refined at 2.7 A resolution to an R-factor of 20.9%. The overall fold of the molecule is essentially the same as yeast pyrophosphatase, except that yeast pyrophosphatase is longer at both the N- and C-termini. Escherichia coli pyrophosphatase is a mixed alpha + beta protein with a complicated topology. The active site cavity, which is also very similar to the yeast enzyme, is formed by seven beta-strands and an alpha-helix and has a rather asymmetric distribution of charged residues. Our structure-based alignment extends and improves upon earlier sequence alignment studies; it shows that probably no more than 14, not 15-17 charged and polar residues are part of the conserved enzyme mechanism of pyrophosphatases. Six of these conserved residues, at the bottom of the active site cavity, form a tight group centred on Asp70 and probably bind the two essential Mg2+ ions. The others, more spreadout and more positively charged, presumably bind substrate. Escherichia coli pyrophosphatase has an extra aspartate residue in the active site cavity, which may explain why the two enzymes bind divalent cation differently. Based on the structure, we have identified a sequence motif that seems to occur only in soluble inorganic pyrophosphatases.
Amino Acid Sequence↗
Analysis of 40 known cystic fibrosis mutations in South African patients.
A total of 140 South African (SA) Caucasoid cystic fibrosis (CF) families were analysed for the common CF mutation, delta F508. The 52 non-delta F508 CF chromosomes in a subset of 127 of these families were also tested for 39 other known CF mutations. The most frequent mutation, apart from delta F508 (which occurs at a frequency of 79%), was G542X (1.3%). Four other mutations, R553X, S549N, 621 + 1G-->T and N1303K, were each found in single families. The other 35 mutations remained unidentified in this sample of CF families. Since 83% of SA Caucasoid CF mutations have been identified, diagnosis by mutation analysis will be possible in only 69% of CF cases. When a diagnosis has been confirmed by a positive sweat test, a combination of linked marker analysis and mutation detection will be necessary if prenatal diagnosis and carrier detection are to be offered in the remaining families.
Cystic Fibrosis↗
Absence of myotonic dystrophy in southern African Negroids is associated with a significantly lower number of CTG trinucleotide repeats.
Myotonic dystrophy (DM) is associated with an increased number of CTG repeats in the 3' untranslated region of the myotonin gene. Because DM has not been observed in southern African Negroids, a study of the CTG repeat polymorphism in this population was undertake. A total of 210 unrelated subjects was studied by PCR analysis of the trinucleotide repeat in the DM gene and the size and distribution of the CTG repeat were determined. The alleles ranged in length from five to 22 repeats. A previously undescribed BglI polymorphism was found which could lead to erroneous diagnosis of DM in people from this population. South African Negroids were found to have significantly fewer large repeat lengths than do white and Japanese populations. It is suggested that the occurrence of fewer large CTG repeats in the normal range may, in part, explain the absence of DM in southern African Negroids.
Africa, Southern↗
Structural repeats in cyclosporin A.
The conformation of various dipeptides of free and cyclophilin A-bound cyclosporin A were compared. Cyclosporin A was shown to contain in both states conformationally duplicated fragments in the putative binding sites for cyclophilin A and phosphatase calcineurin.
Amino Acid Isomerases↗