The Ighmbp2 helicase structure reveals the molecular basis for disease-causing mutations in DMSA1

Mutations in immunoglobulin µ-binding protein 2 (Ighmbp2) cause distal spinal muscular atrophy type 1 (DSMA1), an autosomal recessive disease that is clinically characterized by distal limb weakness and respiratory distress. However, despite extensive studies, the mechanism of disease-causing mutations remains elusive. Here we report the crystal structures of the Ighmbp2 helicase core with and without bound RNA. The structures show that the overall fold of Ighmbp2 is very similar to that of Upf1, a key helicase involved in nonsense-mediated mRNA decay. Similar to Upf1, domains 1B and 1C of Ighmbp2 undergo large conformational changes in response to RNA binding, rotating 30° and 10°, respectively. The RNA binding and ATPase activities of Ighmbp2 are further enhanced by the R3H domain, located just downstream of the helicase core. Mapping of the pathogenic mutations of DSMA1 onto the helicase core structure provides a molecular basis for understanding the disease-causing consequences of Ighmbp2 mutations.     

Analysis of the structure and expression of the c-myc oncogene in cervical tumor and in cervical tumor-derived cell lines

The structure of the c-myc oncogene in 17 cervical tumors and patient-matched nontumor tissues from Chinese patients residing in Taiwan was analysed. In contrast to recent reports on Mexican patients, none of the samples showed rearrangements and sequence amplification in the c-myc gene. The discrepancy may be explained by different carcinogenesis mechanisms being in operation in different geographic regions. Although no structural alterations in the c-myc gene were found in seven cervical carcinoma cell lines analysed, Northern blot analysis indicated different levels of c-myc gene expression which may be related to the presence of human papillomavirus (HPV) sequence in the cell and suggests a possible c-myc-hpv interaction in some stages of the transformation process.     

Structural analysis of an HLA-B27 population variant, B27f. Multiple patterns of amino acid changes within a single polypeptide segment generate polymorphism in HLA-B27

The structure of a new HLA-B27 variant, B27f, distinguishable from other HLA-B27 subtypes by isoelectric focusing and serologic criteria, has been established by comparative peptide mapping and radiochemical sequence analysis. HLA-B27f differs from the major B27.1 subtype in three clustered amino acid replacements: Asp74, Asp77, and Leu81 in B27.1 are changed to Tyr74, Asn77, and Ala81, respectively in B27f. This pattern of differences is analogous to that of HLA-B27.2 in that this subtype also differs from B27.1 in multiple clustered substitutions within the same segment. Thus, polymorphism within the HLA-B27 system is being achieved by introducing different sets of amino acid changes within a particular short segment of the alpha 1 domain. The most likely mechanism for the introduction of multiple changes within this segment is a nonreciprocal recombination event, such as gene conversion. The structural analogies and ethnic distribution of B27f and B27.2 as compared with those of B27.3, and B27.4 support a dynamic model of HLA-B27 evolution in which polymorphism has been created after the separation of the major ethnic groups. In this model, a Caucasian branch would be characterized by subtypes differing from B27.1 in a few changes within the alpha 1 domain, which were probably generated by single genetic steps. An Oriental branch would include those subtypes which differ from B27.1 by changes in both alpha 1 and alpha 2, involving multiple genetic steps for their generation.     

The kinetics and mechanism of shear inactivation of lipase from Candida cylindracea

Shearing experiments were conducted in a stirred tank reactor with 0.1% lipase solutions of Candida cylindracea. Inactivation of the lipase solutions were observed at various shear rates from 50 to 150 s(-1) after continuous shearing for ca. 30-240 min under optimal pH and temperature conditions. However, there was no shear stress denaturation of the lipase when it was subjected to shear stresses of 0.72-109.2 kg/m/s(2) and shear rate of 100 s(-1). In the presence of polypropylene glycol, the rate of denaturation of the lipase decreased by 93%. When the lipase solution was filled to the brim, the rate of denaturation of the lipase decreased by 97% compared to that when reactor was half-filled. The rate of denaturation of the lipase decreased by 61% when probes in the fermentor were removed. There was no significant difference in the rate of denaturation of the lipase under ambient conditions compared with that in the absence of oxygen, or in the absence of free metal ions. Recovery of lipase activity from the first hour of shearing was observed at a shear rate of 150 s(-1). The native lipase and the lipase which had recovered its activity showed similar pH profiles, temperature profiles, and activation energies. Temperature was found to have no effect in the rate of shear-induced denaturation of the lipase in the range 20 to 30 degrees C during shearing at 100 s (-1)and optimal pH. Above 30 degrees C, the rate of denaturation of the lipase increased drastically as a function of temperature. The significance of the findings in the de sign of reactor systems for hydrolysis or esterification of oils by lipase will be discussed.     

Mitochondrial H+-ATPase in mutants of Saccharomyces cerevisiae with defective subunit 8 of the enzyme complex

Mutants of Saccharomyces cerevisiae carrying defined lesions in the mitochondrial aap1 gene, coding for membrane subunit 8 of the H+-ATPase, have been investigated to examine the consequence of the mutations on the function and assembly of the enzyme complex. These include three mit- mutants, which cannot grow by oxidative metabolism due to their inability to synthesize full-length subunit 8, and three partial revertants of one of the mutants. The mutations in these strains have been previously characterized by DNA sequencing. The use of a monoclonal antibody to the beta subunit of the H+-ATPase as a probe of assembly defect revealed that the presence of subunit 8 is essential for the assembly of subunit 6 to the enzyme complex. Mitochondria isolated from the mit- mutants have negligible [32Pi]ATP exchange activity and they exhibited ATPase activity which is not sensitive to inhibition by oligomycin, indicating a defective membrane F0 sector. Normal assembly of subunit 8 (and subunit 6) was observed in the revertant strains, despite 8-9 amino-acid substitutions in the membrane-spanning region of the H+-ATPase subunit 8 in two of the strains. The assembled complex, however, exhibited reduced [32Pi]ATP exchange activity and low sensitivity to oligomycin, indicating that the product of the aap1 gene is a functional subunit of the mitochondrial H+-ATPase.     

Failure of gallamine and pancuronium to inhibit selectively (-)-[3H]quinuclidinyl benzilate binding in guinea-pig atria

Binding of (-)-[3H]quinuclidinyl benzilate (QNB) to muscarinic sites in guinea-pig atrial and ileal longitudinal muscle homogenates showed the presence of a single population of binding sites in atria (KD = 41 (32-53) (95% confidence limits) pM; Bmax = 0.225 +/- 0.02 pmol/mg protein (3)) and two binding sites in the ileum (KD = 20.9 (8.8-49) pM and 11.3 nM; Bmax = 0.436 +/- 0.09 and 11.85 +/- 2.63 pmol/mg protein (4), respectively). Atropine, gallamine, and pancuronium displaced (-)-[3H]QNB binding from the high affinity binding sites in the two tissues in a dose-dependent manner with -log Ki values of 8.6, 6.4, and 6.9, respectively, in atria and 8.7, 6.8, and 6.9, respectively, in ileal longitudinal muscle. The lack of selectivity of gallamine and pancuronium in binding experiments differed from results obtained in isolated tissue experiments where these antagonists showed a marked difference in their ability to antagonize cholinomimetics in the two tissues. In addition, the Ki values for gallamine and pancuronium in ileal homogenates were ca. 130- and 16-fold lower, respectively, than their KB values determined from isolated tissue experiments. Attempts to correlate data from binding experiments and isolated tissue experiments using combinations of antagonists led to variable results attributed to differences in the rates of dissociation of the antagonists from muscarinic receptors. It is concluded that the interaction of gallamine or pancuronium with agonists or antagonists at muscarinic receptors is not a simple bimolecular interaction.     

Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.     

Molecular characterization of de novo secondary trisomy 13.

Unbalanced Robertsonian translocations are a significant cause of mental retardation and fetal wastage. The majority of homologous rearrangements of chromosome 21 in Down syndrome have been shown to be isochromosomes. Aside from chromosome 21, very little is known about other acrocentric homologous rearrangements. In this study, four cases of de novo secondary trisomy 13 are presented. FISH using alpha-satellite sequences, rDNA, and a pTRI-6 satellite I sequence specific to the short arm of chromosome 13 showed all four rearrangements to be dicentric and apparently devoid of ribosomal genes. Three of four rearrangements retained the pTRI-6 satellite I sequence. Case 1 was the exception, showing a deletion of this sequence in the rearrangement, although both parental chromosomes 13 had strong positive hybridization signals. Eleven microsatellite markers from chromosome 13 were also used to characterize the rearrangements. Of the four possible outcomes, one maternal Robertsonian translocation, two paternal isochromosomes, and one maternal isochromosome were observed. A double recombination was observed in the maternally derived rob(13q13q). No recombination events were detected in any isochromosome. The parental origins and molecular chromosomal structure of these cases are compared with previous studies of de novo acrocentric rearrangements.