Expanding the phenotype of DNAJC3 mutations: A case with hypothyroidism additionally to diabetes mellitus and multisystemic neurodegeneration.

Identification of this additional patient from a distant part of the originally described pedigree (Synofzik et al. 2014) confirms pathogenicity of DNAJC3 mutations. Hypothyroidism is a newly identified feature in addition to the known phenotype (diabetes with multisystemic neurodegeneration).

Can color-coded parametric maps improve dynamic enhancement pattern analysis in MR mammography?

PURPOSE: Post-contrast enhancement characteristics (PEC) are a major criterion for differential diagnosis in MR mammography (MRM). Manual placement of regions of interest (ROIs) to obtain time/signal intensity curves (TSIC) is the standard approach to assess dynamic enhancement data. Computers can automatically calculate the TSIC in every lesion voxel and combine this data to form one color-coded parametric map (CCPM). Thus, the TSIC of the whole lesion can be assessed. This investigation was conducted to compare the diagnostic accuracy (DA) of CCPM with TSIC for the assessment of PEC. MATERIALS AND METHODS: 329 consecutive patients with 469 histologically verified lesions were examined. MRM was performed according to a standard protocol (1.5 T, 0.1 mmol/kgbw Gd-DTPA). ROIs were drawn manually within any lesion to calculate the TSIC. CCPMs were created in all patients using dedicated software (CAD Sciences). Both methods were rated by 2 observers in consensus on an ordinal scale. Receiver operating characteristics (ROC) analysis was used to compare both methods. RESULTS: The area under the curve (AUC) was significantly (p=0.026) higher for CCPM (0.829) than TSIC (0.749). The sensitivity was 88.5% (CCPM) vs. 82.8% (TSIC), whereas equal specificity levels were found (CCPM: 63.7%, TSIC: 63.0%). CONCLUSION: The color-coded parametric maps (CCPMs) showed a significantly higher DA compared to TSIC, in particular the sensitivity could be increased. Therefore, the CCPM method is a feasible approach to assessing dynamic data in MRM and condenses several imaging series into one parametric map.

Novel bacterial acetyl coenzyme A carboxylase inhibitors with antibiotic efficacy in vivo.

The pseudopeptide pyrrolidinedione antibiotics, such as moiramide B, have recently been discovered to target the multisubunit acetyl coenzyme A (acetyl-CoA) carboxylases of bacteria. In this paper, we describe synthetic variations of each moiety of the modularly composed pyrrolidinediones, providing insight into structure-activity relationships of biochemical target activity, in vitro potency, and in vivo efficacy. The novel derivatives showed highly improved activities against gram-positive bacteria compared to those of previously reported variants. The compounds exhibited a MIC(90) value of 0.1 microg/ml against a broad spectrum of Staphylococcus aureus clinical isolates. No cross-resistance to antibiotics currently used in clinical practice was observed. Resistance mutations induced by pyrrolidinediones are exclusively located in the carboxyltransferase subunits of the bacterial acetyl-CoA carboxylase, indicating the identical mechanisms of action of all derivatives tested. Improvement of the physicochemical profile was achieved by salt formation, leading to aqueous solubilities of up to 5 g/liter. For the first time, the in vitro activity of this compound class was compared with its in vivo efficacy, demonstrating a path from compounds weakly active in vivo to agents with significant efficacy. In a murine model of S. aureus sepsis, the 100% effective dose of the best compound reported was 25 mg/kg of body weight, only fourfold higher than that of the comparator molecule linezolid. The obvious improvements achieved by chemical derivatization reflect the potential of this novel antibiotic compound class for future therapy.

Discovering the mechanism of action of novel antibacterial agents through transcriptional profiling of conditional mutants.

We present a new strategy for predicting novel antibiotic mechanisms of action based on the analysis of whole-genome microarray data. We first built up a reference compendium of Bacillus subtilis expression profiles induced by 14 different antibiotics. This data set was expanded by adding expression profiles from mutants that showed downregulation of genes coding for proven or emerging antibacterial targets. Here, we investigate conditional mutants underexpressing ileS, pheST, fabF, and accDA, each of which is essential for growth. Our proof-of-principle analyses reveal that conditional mutants can be used to mimic chemical inhibition of the corresponding gene products. Moreover, we show that a statistical data analysis combined with thorough pathway and regulon analysis can pinpoint the molecular target of uncharacterized antibiotics. We apply this approach to two novel antibiotics: a recently published phenyl-thiazolylurea derivative and the natural product moiramide B. Our results support recent findings suggesting that the phenyl-thiazolylurea derivative is a novel phenylalanyl-tRNA synthetase inhibitor. Finally, we propose a completely novel antibiotic mechanism of action for moiramide B based on inhibition of the bacterial acetyl coenzyme A carboxylase.

Effective aromatase inhibition by anastrozole in a patient with gonadotropin-independent precocious puberty in McCune-Albright syndrome.

Testolactone is used to treat conditions with excessive estrogen synthesis, e.g. gonadotropin-independent precocious puberty in McCune-Albright syndrome (MAS). Unfortunately, daily treatment with testolactone requires 3 to 4 doses (10-20 tablets) and even at these doses it is sometimes ineffective. We treated a patient with MAS (café-au-lait spots; thelarche at age 2- 6/12 yr; menarche at 5- 5/12 yr; accelerated bone age [BA 10 yr]) with the highly selective aromatase inhibitor anastrozole (1 mg once per day). Tamoxifen 1 mg/kg per day was added for 1 year but was discontinued when an ovarian cyst developed with markedly elevated estradiol levels. Estradiol levels returned to normal after resuming anastrozole-only treatment and accelerated BA progressed only 6 months during 2 1/2 years of treatment. The potent estrogen suppressive action and simple dosage regimen of anastrozole suggest it may be advantageous compared to other aromatase inhibitors such as testolactone or anti-estrogens.

Identification of novel essential Escherichia coli genes conserved among pathogenic bacteria.

We deleted a subset of 27 open reading frames (ORFs) from Escherichia coli which encode previously uncharacterized, probably soluble gene products homologous to proteins from a broad spectrum of bacterial pathogens such as Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae and Enterococcus faecalis and only distantly related to eukaryotic proteins. Six novel bacteria-specific genes essential for growth in complex medium could be identified through a combination of bioinformatics-based and experimental approaches. We also compared our data to published results of gene inactivation projects with Mycoplasma genitalium and Bacillus subtilis and looked for homologs in all known prokaryotic genomes. Such analyses highlight the enormous metabolic flexibility of prokaryotes. Six of 27 studied genes have been functionally characterized up to now, amongst these four of the essential genes. The gene products YgbP, YgbB and YchB are involved in the non-mevalonate pathway of isoprenoid biosynthesis. KdtB is characterized as the posphopantetheine adenylyltransferase CoaD. There are indications that the other two essential gene products YjeE and YqgF, which we have identified, also possess enzymatic functions. These findings demonstrate the potential of such proteins to be used in screening of large chemical libraries for inhibitors which could be further developed to novel broad-spectrum antibiotics.

High-resolution transcriptional analysis of the symbiotic plasmid of Rhizobium sp. NGR234.

Most of the bacterial genes involved in nodulation of legumes (nod, nol and noe ) as well as nitrogen fixation (nif and fix ) are carried on pNGR234a, the 536 kb symbiotic plasmid (pSym) of the broad-host-range Rhizobium sp. NGR234. Putative transcription regulators comprise 24 of the predicted 416 open reading frames (ORFs) contained on this replicon. Computational analyses identified 19 nod boxes and 16 conserved NifA-sigma54 regulatory sequences, which are thought to co-ordinate the expression of nodulation and nitrogen fixation genes respectively. To analyse transcription of all putative ORFs, the nucleotide sequence of pNGR234a was divided into 441 segments designed to represent all coding and intergenic regions. Each of these segments was amplified by polymerase chain reactions, transferred to filters and probed with radioactively labelled RNA. RNA was extracted from bacterial cultures grown under various experimental conditions, as well as from bacteroids of determinate and indeterminate nodules. Generally, genes involved in the synthesis of Nod factors (e.g. the three hsn loci) were induced rapidly after the addition of flavonoids, whereas others thought to act within the plant (e.g. those encoding the type III secretion system) responded more slowly. Many insertion (IS) and transposon (Tn)-like sequences were expressed strongly under all conditions tested, while a number of loci other than those known to encode nod, noe, nol, nif and fix genes were also transcribed in nodules. Many more diverse transcripts were found in bacteroids of determinate as opposed to indeterminate nodules.

nodD2 of Rhizobium sp. NGR234 is involved in the repression of the nodABC operon.

Transcriptional regulators of the lysR family largely control the expression of bacterial symbiotic genes. Rhizobium sp. NGR234 contains at least four members of this family: two resemble nodD, while two others are more closely related to syrM. Part of the extremely broad host range of NGR234 can be attributed to nodD1, although the second gene shares a high degree of DNA sequence homology with nodD2 of R. fredii USDA191. A nodD2 mutant of NGR234 was constructed by insertional mutagenesis. This mutant (NGR omega nodD2) was deficient in nitrogen fixation on Vigna unguiculata and induced pseudonodules on Tephrosia vogelii. Several other host plants were tested, but no correlation could be drawn between the phenotype and nodule morphology. Moreover, nodD2 has a negative effect on the production of Nod factors: mutation of this gene results in a fivefold increase in Nod factor production. Surprisingly, while the structure of Nod factors from free-living cultures of NGR omega nodD2 remained unchanged, those from V. unguiculata nodules induced by the same strain are non-fucosylated and have a lower degree of oligomerization. In other words, developmental regulation of Nod factor production is also abolished in this mutant. Competitive RNA hybridizations, gene fusions and mobility shift assays confirmed that nodD2 downregulates expression of the nodABC operon.

Structure and evolution of NGRRS-1, a complex, repeated element in the genome of Rhizobium sp. strain NGR234.

Much of the remarkable ability of Rhizobium sp. strain NGR234 to nodulate at least 110 genera of legumes, as well as the nonlegume Parasponia andersonii, stems from the more than 80 different Nod factors it secretes. Except for nodE, nodG, and nodPQ, which are on the chromosome, most Nod factor biosynthesis genes are dispersed over the 536,165-bp symbiotic plasmid, pNGR234a. Mosaic sequences and insertion sequences (ISs) comprise 18% of pNGR234a. Many of them are clustered, and these IS islands divide the replicon into large blocks of functionally related genes. At 6 kb, NGRRS-1 is a striking example: there is one copy on pNGR234a and three others on the chromosome. DNA sequence comparisons of two NGRRS-1 elements identified three types of IS, NGRIS-2, NGRIS-4, and NGRIS-10. Here we show that all four copies of NGRRS-1 probably originated from transposition of NGRIS-4 into a more ancient IS-like sequence, NGRIS-10. Remarkably, all nine copies of NGRIS-4 have transposed into other ISs. It is unclear whether the accumulation of potentially mutagenic sequences in large clusters is due to the nature of the IS involved or to some selection process. Nevertheless, a direct consequence of the preferential targeting of transposons into such IS islands is to minimize the likelihood of disrupting vital functions.

Sulphation of Rhizobium sp. NGR234 Nod factors is dependent on noeE, a new host-specificity gene.

Rhizobia secrete specific lipo-chitooligosaccharide signals (LCOs) called Nod factors that are required for infection and nodulation of legumes. In Rhizobium sp. NGR234, the reducing N-acetyl-D-glucosamine of LCOs is substituted at C6 with 2-O-methyl-L-fucose which can be acetylated or sulphated. We identified a flavonoid-inducible locus on the symbiotic plasmid pNGR234a that contains a new nodulation gene, noeE, which is required for the sulphation of NGR234 Nod factors (NodNGR). noeE was identified by conjugation into the closely related Rhizobium fredii strain USDA257, which produces fucosylated but non-sulphated Nod factors (NodUSDA). R. fredii transconjugants producing sulphated LCOs acquire the capacity to nodulate Calopogonium caeruleum. Furthermore, mutation of noeE (NGRdelta noeE) abolishes the production of sulphated LCOs and prevents nodulation of Pachyrhizus tuberosus. The sulphotransferase activity linked to NoeE is specific for fucose. In contrast, the sulphotransferase NodH of Rhizobium meliloti seems to be less specific than NoeE, because its introduction into NGRdelta noeE leads to the production of a mixture of LCOs that are sulphated on C6 of the reducing terminus and sulphated on the 2-O-methylfucose residue. Together, these findings show that noeE is a host-specificity gene which probably encodes a fucose-specific sulphotransferase.

Molecular basis of symbiosis between Rhizobium and legumes.

Access to mineral nitrogen often limits plant growth, and so symbiotic relationships have evolved between plants and a variety of nitrogen-fixing organisms. These associations are responsible for reducing 120 million tonnes of atmospheric nitrogen to ammonia each year. In agriculture, independence from nitrogenous fertilizers expands crop production and minimizes pollution of water tables, lakes and rivers. Here we present the complete nucleotide sequence and gene complement of the plasmid from Rhizobium sp. NGR234 that endows the bacterium with the ability to associate symbiotically with leguminous plants. In conjunction with transcriptional analyses, these data demonstrate the presence of new symbiotic loci and signalling mechanisms. The sequence and organization of genes involved in replication and conjugal transfer are similar to those of Agrobacterium, suggesting a recent lateral transfer of genetic information.

Subunit structure and organization of the genes of the A1A0 ATPase from the Archaeon Methanosarcina mazei Gö1.

The proton-translocating A1A0 ATP synthase/hydrolase of Methanosarcina mazei Gö1 was purified and shown to consist of six subunits of molecular masses of 65, 49, 40, 36, 25, and 7 kDa. Electron microscopy revealed that this enzyme is organized in two domains, the hydrophilic A1 and the hydrophobic A0 domain, which are connected by a stalk. Genes coding for seven hydrophilic subunits were cloned and sequenced. From these data it is evident that the 65-, 49-, 40- and 25-kDa subunits are encoded by ahaA, ahaB, ahaC, and ahaD, respectively; they are part of the A1 domain or the stalk. In addition there are three more genes, ahaE, ahaF, and ahaG, encoding hydrophilic subunits, which were apparently lost during the purification of the protein. The A0 domain consists of at least the 7-kDa proteolipid and the 36-kDa subunit for which the genes have not yet been found. In summary, it is proposed that the A1A0 ATPase of Methanosarcina mazei Gö1 contains at least nine subunits, of which seven are located in A1 and/or the stalk and two in A0.

Sequencing the 500-kb GC-rich symbiotic replicon of Rhizobium sp. NGR234 using dye terminators and a thermostable "sequenase": a beginning.

Genomes of the soil-borne nitrogen-fixing symbionts of legumes [Azo(Brady)Rhizobium species] typically have GC contents of 59-65 mol%. As a consequence, compressions (up to 400 per cosmid) are common using automated dye primer shotgun sequencing methods. To overcome this difficulty, we have exclusively applied dye terminators in combination with a thermostable "sequenase" for shotgun sequencing GC-rich cosmids from pNGR234a, the 500-kbp symbiotic replicon of Rhizobium sp. NGR234. A thermostable sequenase incorporates dye terminators into DNA more efficiently than Taq DNA polymerase, thus reducing the concentrations needed (20- to 250-fold). Unincorporated dye terminators can simply be removed by ethanol precipitation. Here, we present data of pXB296, one of 23 overlapping cosmids representing pNGR234a. We demonstrate that the greatly reduced number of compressions results in a much faster assembly of cosmid sequence data by comparing assembly of the shotgun data from pXB296 and the data from another pNGR234a cosmid (pXB110) sequenced using dye primer methods. Within the 34,010-bp sequence from pXB296, 28 coding regions were predicted. All of them showed significant homologies to known proteins, including oligopeptide permeases, an essential cluster for nitrogen fixation, and the C4-dicarboxylate transporter DctA.

[Doppler studies of arterial uterofetoplacental blood flow before and during labor]. / Doppleruntersuchungen des arteriellen utero-feto-plazentaren Blutflusses vor und während der Geburt.

The encouraging results obtained using pulsed Doppler sonography for antepartal diagnosis gave cause to use the method during labor. For this purpose a group with normal course of pregnancy was examined by Doppler sonography. This group was compared with a similar group examined by the same method at onset of labor with the cervix beginning to dilate, or with premature rupture. A comparison of the usual Doppler parameters, uterine arteries, umbilical artery and fetal aorta, recorded in contraction-free phases, showed no differences between the two groups. A third group was examined by Doppler sonography during labor with average or late cervix dilatation. In this case the Doppler parameters for the contraction phases were compared with those for the contraction-free phases. With adequate utero-placental supply during labor, the changes in the Doppler parameters for the uterine arteries due to contraction indicate a reduction in blood flow. The blood flow in the umbilical artery remains unaffected during normal labor. In the fetal aorta the blood flow velocity drops significantly due to contractions, while the peripheral resistance is unchanged. The elimination of the end-diastolic shift in frequency in the fetal aorta during labor indicates a fetal supply deficiency, as shown by Doppler measurements during birth in cases with pathologic cardiotokograms.