Validation of semiempirical methods for modeling of corrinoid systems

Several semiempirical methods (MNDO-d, PM3tm, PM3-d, PM5, PM6, and AM1-d) have been tested against experimental data and density functional theory (DFT) results in search for the best methods that can be used for quantum-mechanical-molecular mechanics (QM/MM) modeling of corrinoid systems of vitamin B(12) co-factor. It has been found that the PM6 parametrization in its present form gives results closest to hybrid DFT calculations that are most widely used thus far. In comparison with pure DFT and experimental data the best agreement is obtained for PM3tm parametrization, while PM6 yields slightly worse results. AM1-d yields bad geometry of the corrin moiety. The worst performance was observed for MNDO-d, which has severe problem with position and orientation of the alpha-ligands.     

Primary ciliary dyskinesia: genes, candidate genes and chromosomal regions

Primary ciliary dyskinesia (PCD) is a multisystem disease characterized by recurrent respiratory tract infections, sinusitis, bronchiectasis and male subfertility, associated in about 50% patients with situs inversus totalis (the Kartagener syndrome). The disease phenotype is caused by ultrastructural defects of respiratory cilia and sperm tails. PCD is a heterogenetic disorder, usually inherited as an autosomal recessive trait. So far, mutations in two human genes have been proved to cause the disease. However, the pathogenetics of most PCD cases remains unsolved. In this review, the disease pathomechanism is discussed along with the genes that are or may be involved in the pathogenesis of primary ciliary dyskinesia and the Kartagener syndrome.     

Apparent X-linked primary ciliary dyskinesia associated with retinitis pigmentosa and a hearing loss

Three brothers, one 10-year-old and a pair of 14-year-old dizygotic twins--expressed the classical, early-onset retinitis pigmentosa (RP) with typical ophthalmoscopic findings, night blindness, visual field constricted to 10 degrees and flat ERG response. All three brothers were also diagnosed with primary ciliary dyskinesia (PCD) and had recurrent respiratory infections, chronic sinusitis and bronchiectasis. In all of them, resection of the middle lobe of the right lung was performed. A similar clinical picture of coexisting RP and PCD was noted in the brother of the probands' mother. All probands displayed situs solitus. Consistent with the X-linked mode of RP inheritance, there were also three obligatory female carriers of the disorder in this family: the mother of the affected boys, her mother and a daughter of her brother. In all of them, retinitis pigmentosa "sine pigmento" was found with milder but clinically significant symptoms (mild night blindness, visual field constricted to 30 degrees, and scotopic and photopic ERG responses reduced to 30-60%). No extraocular symptoms were detected in any of the heterozygous female carriers. This family presents an example of two rare phenomena: X-linked dominant retinitis pigmentosa (with milder expression in females) and a rare combination of RP with recurrent respiratory infections due to PCD.     

Crystal structure of the hypothetical protein TA1238 from Thermoplasma acidophilum: a new type of helical super-bundle

The crystal structure of the hypothetical protein TA1238 from Thermoplasma acidophilum was solved with multiple-wavelength anomalous diffraction and refined at 2.0 A resolution. The molecule consists of a typical four-helix antiparallel bundle with overhand connection. However, its oligomerization into a trimer leads to a coiled "super-helix" which is novel for such bundles. Its central feature, a six-stranded coiled coil, is also novel for proteins. TA1238 does not have strong sequence homologues in databases, but shows strong structural similarity with some proteins in the Protein Data Bank. The function could not be inferred from the sequence but the structure, with some rearrangement, bears some resemblance to the active site region of cobalamin adenosyltransferase (TA1434). Specifically, TA1238 retains Arg104, which is structurally equivalent to functionally critical Arg119 of TA1434. For such conformational change, the overhand connection of TA1238 might need to be involved in a gating mechanism that might be modulated by ligands and/or by interactions with the physiological partners. This allowed us to hypothesize that TA1238 could be involved in cobalamin biosyntheses.     

Protective effect of vaccination with DNA of the H. Pylori genomic library in experimentally infected mice

Immunologically mediated protection against H. pylori infection is an attractive alternative to antibiotic treatment. We compared the efficacy of conventional protein vaccination with that of genetic vaccination against experimental infection with H. pylori in mice. For oral immunization, we used the recombinant peptide of an antigenic fragment of UreB (rUreB) or H. pylori-whole cell lysate antigens, and for genetic immunization, we used recombinant pcDNA and pSec plasmids inserted with the fragment of ureB or DNA of the H. pylori genomic library. Mice were challenged with the mouse stomach-adapted H. pylori Sidney Strain. The detection of gastric bacterial colonization was performed by real-time PCR of a 26-kDa Helicobacter-specific gene, and the presence of serum H. pylori-specific antibodies was determined using direct ELISA assay. The most effective treatment appeared to be oral vaccination with rUreB and either intramuscular or intradermal vaccination with DNA of the H. pylori genomic library. Intradermal genetic vaccination with genomic library DNA significantly increased the IgG antibody response. Our study revealed acceptable efficacies of genetic vaccination with DNA of the H. pylori genomic library.     

Crystal structure of the putative adapter protein MTH1859

MTH1859 from Methanobacterium thermoautotrophicum is a 77 residue protein representing a conserved family of functionally uncharacterized proteins. We solved the crystal structure of MTH1859 by single wavelength anomalous diffraction phasing using selenomethionine labeled protein. MTH1859 adopts a mainly anti-parallel all-beta-fold. The beta-sheet is heavily bent to form a U-structure that is closed through a loop. The monomer structure possesses similarities to the photoreaction center (PRC) domain fold, but the protein employs a unique oligomerization scheme. Two monomers of MTH1859 occupy the asymmetric unit and dimerize in a head-to-head fashion. Crystal packing interactions identify a second protein-protein interaction interface at the MTH1859 tails which can simultaneously bind two partner molecules. These interactions lead to the formation of a honeycomb structure and suggest that the family of MTH1859-like proteins might function as adapters for protein complex assembly.     

Molecular phylogeny of Demospongiae: implications for classification and scenarios of character evolution

An analysis of the phylogenetic relationships of the 13 orders of Demospongiae, based on 18S and C1, D1 and C2 domains of 28S rRNA (for, respectively, 26 and 32 taxa) has been performed. The class Demospongiae as traditionally defined is not found to be monophyletic. Instead, a clade comprising all demosponges except Homoscleromorpha is well-supported, and we define phylogenetically the name Demospongiae in this more restricted sense to preclude the possibility of drastic alterations of the meaning of Demospongiae in the future, depending on the position of Homoscleromorpha. Within this clade Demospongiae s.s., ceractinomorphs and tetractinomorphs are polyphyletic, implying homoplastic evolution of characters such as reproductive strategies (viviparity vs. oviparity) and skeleton architecture (reticulate vs. radiate). The topology derived from our molecular data provides a basis for proposing a new classification of Demospongiae s.s., and suggests a reverse polarity of some characters, with respect to traditional conceptions: viviparity, presence of monaxon spicules and of spongin appear to be ancestral, whereas oviparity, and presence of tetraxon spicules appear as derived characters.     

A real-time polymerase chain reaction assay for quantification of allele ratios and correction of amplification bias

Allele-specific epigenetic modifications are crucial for several important biological functions, including genomic imprinting and X-inactivation in mammals. Consequently, an ever increasing number of investigations requires accurate quantification of the relative abundance of parental alleles of a specific sequence in a DNA sample. Here, combining the use of polymorphic restriction sites with real-time polymerase chain reaction (PCR) amplification, we describe a simple and quantitative assay to measure allele ratios. The efficiency of the assay was assessed on genomic DNA for several polymorphic restriction sites located in the mouse Igf2/H19 imprinted locus. The assay was also successfully applied to quantify allele ratio in cDNA samples. In addition, we provide an experimental procedure for detection and correction of potential PCR amplification bias which significantly extends the range of application of the assay.     

Low sequence similarity and gene content of symbiotic clusters of Bradyrhizobium sp. WM9 (Lupinus) indicate early divergence of “lupin” lineage in the genus Bradyrhizobium

Two sequenced nodulation regions of lupin Bradyrhizobium sp. WM9 carried the majority of genes involved in the Nod factor production. The nod region I harbored: nolA, nodD, nodA, nodB, nodC, nodS, nodI, nodJ, nolO, nodZ, fixR, nifA, fixA, nodM, nolK and noeL. This gene arrangement resembled that found in the nodulation region of Bradyrhizobium japonicum USDA110, however strain WM9 harbored only one nodD gene copy, while the nodM, nolK and noeL genes had no counterparts in the 410 kb symbiotic region of strain USDA110. Region II harbored nolL and nodW, but lacked an nodV gene. Both regions carried ORFs that lacked similarity to the published USDA110 sequences, though they had homologues in symbiotic regions of Rhizobium etli, Sinorhizobium sp. NGR234 and Mesorhizobium loti. These differences in gene content, as well as a low average sequence identity (70%) of symbiotic genes with respect to B. japonicum USDA110 were in contrast with the phylogenetic relationship of USDA110 and WM9 revealed by the analysis of 16S rDNA and dnaK sequences. This most likely reflected an early divergence of symbiotic loci, and possible co-speciation with distinct legumes. During this process the loss of a noeI gene and the acquisition of a nolL gene could be regarded as an adaptation towards these legumes that responded to Nod factors carrying 4-O-acetylfucose rather than 2-O-methylfucose. This explained various responses of lupins and serradella plants to infection by mutants in nodZ and nolL genes, knowing that serradella is a stringent legume while lupins are more promiscuous legumes. This revised version was published online in August 2006 with corrections to the Cover Date.