FSGS tip lesion in polyautoimmunity including Sjögren's syndrome.

We present a case with five auto-immune phenomena, including Sjögren's syndrome, for which we also diagnosed a tip lesion of focal segmental glomerulosclerosis (FSGS).v About one-third of Sjögren's syndrome patients have renal involvement, but FSGS is rarely reported. FSGS is thought to involve T-cell dysfunction and in this patient with multiple auto-immune phenomena, it may reflect a severe dysregulation of cellular immunity.

Hepatitis C virus transmission in a Dutch haemodialysis unit: detailed outbreak investigation using NS5A gene sequencing.

BACKGROUND: Haemodialysis is a risk factor for hepatitis C virus (HCV) transmission. Two patients receiving haemodialysis in a Dutch dialysis unit in The Hague were found to seroconvert to HCV in December 2016 after the yearly routine control for blood-borne viruses. Following the presumed time of infection, three chronically infected HCV patients were identified as possible index cases. AIM: To confirm inter-patient transmission and to identify the source. METHODS: Molecular investigation and review of medical records were performed. FINDINGS: Both of the incident cases and one of the three possible index cases were demonstrated to be infected with HCV genotype 2b based on 5'UTR sequencing. Epidemiological relatedness between these viruses was further investigated by sequencing of the NS5A region. Phylogenetic analysis clearly identified the incident cases and the index case to represent a cluster distinct from unrelated controls with HCV genotype 2b. Detailed review of the medical records identified two possible incidents that might have resulted in the HCV transmission cases: contamination of the venous pressure-sensing port due to high venous pressures or incomplete compliance with infection control precautions of the unit staff during handling of two incidents, that occurred at the same time in a single haemodialysis session with the index patient as well as both incident cases present. CONCLUSION: This study demonstrates that detailed incident recording in combination with state-of-the-art molecular investigations such as sequencing of the NS5A region resulted in unravelling a set of two HCV transmissions that occurred at a haemodialysis unit.

Regression formulae for ab initio and density functional calculated chemical shifts.

Linear regression formulae are given for converting 1H and 13C magnetic shielding constants calculated at common ab initio and density functional theory levels of calculation into chemical shifts relative to tetramethylsilane. Accuracies of roughly +/-2.2 ppm (13C) and +/-0.15 ppm (1H) or better are found for the training set for most levels. The highest level calculations do not always give better results than economical standard calculations.

Dissected nucleus-independent chemical shift analysis of pi-aromaticity and antiaromaticity.

[structure: see text] Analysis of the basic pi-aromatic (benzene) and antiaromatic (cyclobutadiene) systems by dissected nucleus-independent chemical shifts (NICS) shows the contrasting diatropic and paratropic effects, but also reveals subtleties and unexpected details.

Transcript analysis of multiple copies of amo (encoding ammonia monooxygenase) and hao (encoding hydroxylamine oxidoreductase) in Nitrosomonas europaea.

The genes encoding ammonia monooxygenase (amoCAB), hydroxylamine oxidoreductase (hao), and the c-type cytochrome c-554 (hcy) are present in multiple copies in the genome of Nitrosomonas europaea. The upstream regions of the two copies of amoC, the three copies of hao, and one copy of hcy were cloned and sequenced. Primer extension reactions were done to identify transcription start sites for these genes, as well as for amoA. Putative sigma(70) promoter sequences were found associated with all but one of the mapped transcription start sites. Primer extensions were done with amoC primers using RNA harvested from cells incubated with and without ammonium. The experiments suggested that N. europaea cells may be able to use different promoters in the presence and absence of ammonium.

Differential regulation of amoA and amoB gene copies in Nitrosomonas europaea.

Nitrosomonas europaea contains two nearly identical copies of the operon, amoCAB, which encodes the ammonia monooxygenase (AMO) enzyme. Cells of N. europaea containing single mutations in either amoA or amoB gene copies were incubated in ammonium both prior to and after exposure to acetylene or light. For each strain, the O(2) consumption rates and amounts of AmoA polypeptide, the active site-containing subunit of AMO, produced in each strain were determined. Strains carrying a mutation in either the amoA(2) or amoB(2) genes responded similarly to wild-type cells, but the strains carrying mutations in the amoA(1) or amoB(1) genes responded differently from the wild-type, or from each other. These results suggest that the copies of amoA and amoB are differentially regulated upon exposure to different external stimuli.

Transcription of the amoC, amoA and amoB genes in Nitrosomonas europaea and Nitrosospira sp. NpAV.

Nitrifying bacteria such as Nitrosomonas europaea and Nitrosospira sp. NpAV use ammonia monooxygenase (AMO) for oxidation of their primary growth substrate, ammonia. Two polypeptides of AMO are coded for by contiguous genes, amoA and amoB, which are preceded by a third gene, amoC. The amoCAB clusters are present in multiple copies in nitrifying bacteria of the beta subdivision. These bacteria also have one amoC copy that is not adjacent to a copy of amoAB. The seven known amoC genes in different nitrifiers code for similar polypeptides (> 68%). Reverse transcriptase-polymerase chain reactions and Northern blots indicated that amoC from the amoCAB cluster is contained on a transcript with amoAB. Two other transcripts were detected with amo probes and may be a product of processing of the amoCAB mRNA or independent transcripts.

Mutagenesis and expression of amo, which codes for ammonia monooxygenase in Nitrosomonas europaea.

Nitrosomonas europaea has two copies of the operon encoding ammonia monooxygenase (AMO). The nucleotide sequences of the two copies of amoA were obtained, and they were found to differ by one nucleotide. To determine if both copies of amoA were functional, insertional mutagenesis was performed to inactivate either copy of amoA alone. A DNA cassette containing the lacZ and kan genes inserted into amoA was constructed. Mutagenesis was done by using transformation and homologous recombination to mobilize the cassette into the chromosomal copies of amoA. Mutations were obtained in both copies of amoA. Either copy of amoA was sufficient to support growth when the other copy was disrupted. However, inactivation of one copy of amoA, but not the other, resulted in slower growth. Measurements of ammonia-dependent O2 consumption, which depends on AMO, confirmed that the slower-growing mutant had lower activity while the faster-growing mutant had near wild-type levels of activity. Similarly, as measured by [14C]acetylene label incorporation, there was less active AMO present in the slower-growing mutant than in the faster-growing mutant or in the wild type. Northern blot analysis of transcription likewise showed that the slower-growing mutant had less full-sized AMO mRNA.

Effects of Soil on Ammonia, Ethylene, Chloroethane, and 1,1,1-Trichloroethane Oxidation by Nitrosomonas europaea.

Ammonia monooxygenase (AMO) from Nitrosomonas europaea catalyzes the oxidation of ammonia to hydroxylamine and has been shown to oxidize a variety of halogenated and nonhalogenated hydrocarbons. As part of a program focused upon extending these observations to natural systems, a study was conducted to examine the influence of soil upon the cooxidative abilities of N. europaea. Small quantities of Willamette silt loam (organic carbon content, 1.8%; cation-exchange capacity, 15 cmol/kg of soil) were suspended with N. europaea cells in a soil-slurry-type reaction mixture. The oxidations of ammonia and three different hydrocarbons (ethylene, chloroethane, and 1,1,1-trichloroethane) were compared to results for controls in which no soil was added. The soil significantly inhibited nitrite production from 10 mM ammonium by N. europaea. Inhibition resulted from a combination of ammonium adsorption onto soil colloids and the exchangeable acidity of the soil lowering the pH of the reaction mixture. These phenomena resulted in a substantial drop in the concentration of NH(4) in solution (10 to 4.5 mM) and, depending upon the pH, in a reduction in the amount of available NH(3) to concentrations (8 to 80 muM) similar to the K(s) value of AMO for NH(3) ( approximately 29 muM). At a fixed initial pH (7.8), the presence of soil also modified the rates of oxidation of ethylene and chloroethane and changed the concentrations at which their maximal rates of oxidation occurred. The modifying effects of soil on nitrite production and on the cooxidation of ethylene and chloroethane could be circumvented by raising the ammonium concentration in the reaction mixture from 10 to 50 mM. Soil had virtually no effect on the oxidation of 1,1,1-trichloroethane.

Mutagenesis of hydroxylamine oxidoreductase in Nitrosomonas europaea by transformation and recombination.

Mutagenesis of Nitrosomonas europaea was achieved by electroporation and recombination. To demonstrate this, an aminoglycoside 3'-phosphotransferase (kan) gene was specifically inserted into each of the three gene copies of hao individually. Southern hybridizations and PCR analysis showed the incorporation of the kan gene at the chosen genetic loci. The isolation of mutant strains was achieved in 7 to 14 days when the strains were grown on solid medium. The induced mutations were stable even in the absence of kanamycin-selective pressure for periods of up to 45 days in culture. The mutant strains did not show an observable phenotype different from that of the wild type when grown under the same conditions.

Induction of ammonia monooxygenase and hydroxylamine oxidoreductase mRNAs by ammonium in Nitrosomonas europaea.

In Nitrosomonas europaea, ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) catalyse the oxidation of ammonia (NH3) to nitrite (NO2-). A transcript of 3500 bases hybridizes to probes for amoA and amoB (genes that code for AMO proteins). A transcript of 2100 bases hybridizes to probes for hao (the gene that codes for HAO). Induction of the mRNAs detected by amo and hao probes required the presence of ammonium (NH4+). To correlate new levels of mRNA with de novo activity, existent mRNA pools and AMO activity were depleted prior to induction by NH4+. The mRNAs of AMO and HAO were depleted by depriving the cells of energy for at least 8 h; AMO activity was inactivated with acetylene (C2H2) after mRNA depletion. In cells treated this way, levels of new AMO mRNA and de novo AMO enzyme activity were correlated with increased NH4+ concentrations up to 1 mM after 3 h of incubation. HAO mRNA also increased in the NH4(+)-treated cells. Other proteins and RNAs induced by NH4+ were detected in 14CO2-labelling experiments. The AMO and HAO mRNAs were preferentially synthesized during energy-limiting conditions.

Generation of polymerase chain reaction-specific probes for library screening using single degenerate primers.

Degenerate oligonucleotide primers were made to peptide sequences from hydroxylamine oxidoreductase (HAO) from Nitrosomonas europaea. The primers were used singly in PCR reactions to amplify portions of the gene for HAO from genomic DNA. Southern hybridizations using fragments amplified with each primer showed that they labeled the same genomic DNA fragments. The PCR-amplified fragments were successfully used to screen a gene library for clones containing the HAO gene. The method of isolating genes by PCR with single primers has general utility.

Sequence of hcy, a gene encoding cytochrome c-554 from Nitrosomonas europaea.

Cytochrome c-554 (Cyt c-554) was purified from Nitrosomonas europaea. The N-terminal and internal amino acid sequences were determined. A synthetic oligodeoxyribonucleotide primer based on the N-terminal sequence was used to construct a PCR clone. This clone was used to identify genomic DNA fragments containing the gene encoding Cyt c-554. We determined the nucleotide sequence of this gene and named it hcy for hydroxylamine oxidoreductase-linked cytochrome.

Characterization of the gene encoding hydroxylamine oxidoreductase in Nitrosomonas europaea.

Hydroxylamine oxidoreductase (HAO) catalyzes the oxidation of hydroxylamine to nitrite in Nitrosomonas europaea. The electrons released in the reaction are partitioned to ammonium monooxygenase and to the respiratory chain. The immediate acceptor of electrons from HAO is believed to be cytochrome c-554 (Cyt c-554). We have isolated a genomic DNA fragment containing the structural gene encoding HAO (hao) and a part of the gene for Cyt c-554. The nucleotide sequence of hao was determined, and its transcription was analyzed. The open reading frame (ORF) encodes amino acid sequences matching the purified peptides of HAO. A 64.28-kDa protein is encoded in this ORF, in close agreement with the empirically determined molecular mass of 63 kDa. The N terminus was located 24 amino acids from the start codon, suggesting the presence of a leader sequence. The putative eight heme-binding peptides were localized in this ORF. The gene for Cyt c-554 was located 1,200 bp downstream from the 3' end of hao. An ORF was identified in the upstream region from hao and may encode a protein of unknown function. Data bank searches did not reveal proteins with substantial similarities to HAO, but they did reveal similarities between Cyt c-554 and other c-type cytochromes.

Inducible expression of cytokinin biosynthesis in Agrobacterium tumefaciens by plant phenolics.

Nopaline strains of Agrobacterium tumefaciens contain a gene, tzs, that encodes a cytokinin biosynthetic prenyl transferase. The gene is located adjacent to the Ti plasmid virulence region and is constitutively expressed at low levels. As a result, bacteria containing tzs secrete low levels of zeatin into the medium. We find zeatin secretion to be induced more than 100-fold by acetosyringone, one of a number of naturally occurring phenolics produced by plants in response to wounding. Induction was very sensitive to the pH of the medium (optimum pH 5.5) and was due to massive overexpression of tzs-encoded cytokinin prenyl transferase activity. The relative ability of members of a set of phenols to induce tzs expression was examined and found to be parallel to that reported for activation of other virulence genes. A series of molecular cloning experiments established that virA and virG, two genes known to be essential to the virulence induction process, were necessary and sufficient for phenolic-induced tzs expression. Sequences present in the promoter region of tzs were found to be similar to those present in genes regulated by bacterial two-component positive regulatory systems.