Clinical and biochemical description of a novel CYP21A2 gene mutation 962_963insA using a new 3D model for the P450c21 protein

OBJECTIVE: A severely virilized 46, XX newborn girl was referred to our center for evaluation and treatment of congenital adrenal hyperplasia (CAH) because of highly elevated 17alpha-hydroxyprogesterone levels at newborn screening; biochemical tests confirmed the diagnosis of salt-wasting CAH. Genetic analysis revealed that the girl was compound heterozygote for a previously reported Q318X mutation in exon 8 and a novel insertion of an adenine between nucleotides 962 and 963 in exon 4 of the CYP21A2 gene. This 962_963insA mutation created a frameshift leading to a stop codon at amino acid 161 of the P450c21 protein. AIM AND METHODS: To better understand structure-function relationships of mutant P450c21 proteins, we performed multiple sequence alignments of P450c21 with three mammalian P450s (P450 2C8, 2C9 and 2B4) with known structures as well as with human P450c17. Comparative molecular modeling of human P450c21 was then performed by MODELLER using the X-ray crystal structure of rabbit P450 2B4 as a template. RESULTS: The new three dimensional model of human P450c21 and the sequence alignment were found to be helpful in predicting the role of various amino acids in P450c21, especially those involved in heme binding and interaction with P450 oxidoreductase, the obligate electron donor. CONCLUSION: Our model will help in analyzing the genotype-phenotype relationship of P450c21 mutations which have not been tested for their functional activity in an in vitro assay.

A new variant of Actinobacillus pleuropneumoniae serotype 3 lacking the entire apxII operon

Actinobacillus pleuropneumoniae is an important respiratory pathogen causing pleuropneumonia in pig. The species is genetically characterized by the presence of 4 RTX (Repeats in the Structural ToXin) toxin genes: apxI, apxII, and apxIII genes are differentially present in various combinations among the different serotypes, thereby defining pathogenicity; the apxIV gene is present in all serotypes. Polymerase chain reaction (PCR)-based apx gene typing is done in many veterinary diagnostic laboratories, especially reference laboratories. The present report describes the isolation of atypical A. pleuropneumoniae from 4 independent cases from 2 countries. All isolates were beta-nicotinamide adenine dinucleotide (beta-NAD) dependent and nonhemolytic but showed strong co-hemolysis with the sphingomyelinase of Staphylococcus aureus on sheep blood agar. Classical biochemical tests as well as Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and sequence-based analysis (16S ribosomal RNA [rRNA] and rpoB genes) identified them as A. pleuropneumoniae. Apx-toxin gene typing using 2 different PCR systems showed the presence of apxIV and only the apxIII operon (apxIIICABD). None of the apxI or apxII genes were present as confirmed by Southern blot analysis. The 16S rRNA and rpoB gene analyses as well as serotype-specific PCR indicate that the isolates are variants of serotype 3. Strains harboring only apxIV and the apxIII operon are possibly emerging types of A. pleuropneumoniae and should therefore be carefully monitored for epidemiological reasons.

Streptococcus spp. and related bacteria: Their identification and their pathogenic potential for chronic mastitis - A molecular approach

Streptococcus spp. and related bacteria form a large group of organisms which are associated with bovine intramammary Infections (IMI). Some of them are the well-known mastitis pathogens Streptococcus uberis and Streptococcus agalactiae. In addition, there are a considerable number of these gram-positive, catalase-negative cocci (PNC) with unclear mastitic pathogenicity such as Aerococcus viridans which make the conventional diagnostics of PNC difficult. One diagnostic, API 20 Strep (API, Biomerieux) is recommended which, as a phenotypic assay, involves a series of miniaturized biochemical tests. Recently, preference is given to genotypic identification methods. In particular, sequencing of the 16S rRNA gene allows highly reproducible and accurate identification of bacteria and permits discovery of novel, clinically relevant bacteria. As a consequence, the aim of the present study was to compare identification of IMI-associated PNC by the API method as well as by sequencing of their 16S rRNA gene (16S). Furthermore, the correlation of these bacteria to bovine chronic mastitis and their phylogeny was investigated. 102 PNC isolated from single quarter milk samples were identified by API and 16S sequencing. Considering Streptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae, both methods generated fully concordant results. In contrast, a very high disconcordance was observed for most of the other PNC, in particular Enterococcus spp., Aerococcus viridans and the viridans streptococci were shown as apathogenic. Lactococcus garvieae was found to be an opportunistic pathogen causing IMI during late lactation. In addition, PNC isolated from milk were frequently observed together with other bacteria, in particular with Staphylococcus spp. In these cases, the levels of somatic cell counts (SCC) were determined by the specific PNC present in the sample. Considering PNC phylogeny based on 16S sequencing, 3 major clusters were observed. They included all the common mastitis pathogens (cluster I), the Lactococcus spp., Enterococcus spp. and Aerococcus spp. (cluster II) and all the viridans streptococci (cluster III).

Phenotypic and genetic characterization of Pasteurella multocida and related isolates from rabbits in Switzerland

Several bacteria belonging to the family Pasteurellaceae are potential pathogens in rabbits. In particular, Pasteurella multocida is considered to be important, and outbreaks caused by this species result in considerable economic losses in rabbitries. However, Pasteurellaceae spp. isolated from rabbits are poorly characterized, and thus, proper identification of P. multocida isolates from these animals is problematic and often unsatisfactory, thereby hampering epidemiological investigations. Therefore, 228 isolates from rabbit populations originating from a breeding and fattening organization with group management and postmortem cases with pasteurellosis from individual owners were phenotypically and genotypically analyzed using biochemical tests and repetitive extragenic palindromic polymerase chain reaction (REP-PCR). Furthermore, 41 samples representing observed phenotypes were selected for phylogenetic analysis using 16S ribosomal RNA and rpoB genes. The REP-PCR typing and phylogenetic analyses correlated well and appeared to be distinct molecular methods for characterization of rabbit isolates. Phenotyping, however, diverged from molecular recognition, reflecting the problematic conventional diagnosis of these strains. The fermentation of sorbitol appeared to be an imprecise indicator for P. multocida subspecies classification. According to REP-PCR and sequencing results, 82% of the isolates were characterized as P. multocida subsp. multocida, 3% as P. multocida subsp. septica, and 5% as P. multocida. Further, 5% were identified as Pasteurella canis. The other 5% represented a homogeneous group of unknown species belonging to the Pasteurellaceae. Samples obtained from individual postmortem cases demonstrated a higher phenotypic and genetic heterogeneity than samples from group management rabbits.

Synthesis and properties of isobicyclo-DNA

We present the synthesis of the isobicyclo-DNA building blocks with the nucleobases A, C, G and T, as well as biophysical and biological properties of oligonucleotides derived thereof. The synthesis of the sugar part was achieved in 5 steps starting from a known intermediate of the tricyclo-DNA synthesis. Dodecamers containing single isobicyclo-thymidine incorporations, fully modified A- and T-containing sequences, and fully modified oligonucleotides containing all four bases were synthesized and characterized. Isobicyclo-DNA forms stable duplexes with natural nucleic acids with a pronounced preference for DNA over RNA as complements. The most stable duplexes, however, arise by self-pairing. Isobicyclo-DNA forms preferentially B-DNA-like duplexes with DNA and A-like duplexes with complementary RNA as determined by circular dichroism (CD) spectroscopy. Self-paired duplexes show a yet unknown structure, as judged from CD spectroscopy. Biochemical tests revealed that isobicyclo-DNA is stable in fetal bovine serum and does not elicit RNaseH activity.

Glycoprotein biosynthesis in a eukaryote lacking the membrane protein Rft1

Mature dolichol-linked oligosaccharides (mDLOs) needed for eukaryotic protein N-glycosylation are synthesized by a multistep pathway in which the biosynthetic lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) flips from the cytoplasmic to the luminal face of the endoplasmic reticulum. The endoplasmic reticulum membrane protein Rft1 is intimately involved in mDLO biosynthesis. Yeast genetic analyses implicated Rft1 as the M5-DLO flippase, but because biochemical tests challenged this assignment, the function of Rft1 remains obscure. To understand the role of Rft1, we sought to analyze mDLO biosynthesis in vivo in the complete absence of the protein. Rft1 is essential for yeast viability, and no Rft1-null organisms are currently available. Here, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote whose Rft1 homologue functions in yeast. We report that TbRft1-null procyclic trypanosomes grow nearly normally. They have normal steady-state levels of mDLO and significant N-glycosylation, indicating robust M5-DLO flippase activity. Remarkably, the mutant cells have 30-100-fold greater steady-state levels of M5-DLO than wild-type cells. All N-glycans in the TbRft1-null cells originate from mDLO indicating that the M5-DLO excess is not available for glycosylation. These results suggest that rather than facilitating M5-DLO flipping, Rft1 facilitates conversion of M5-DLO to mDLO by another mechanism, possibly by acting as an M5-DLO chaperone.

Ultrastructural mitochondrial alterations in Equine myopathies of unknown origin.

Background: Very few mitochondrial myopathies have been described in horses. Objective: To examine the ultrastructure of muscle mitochondria in equine cases of myopathy of unknown origin. Materials & methods: Biopsies of vastus lateralis of the Musculus quadriceps femoris were taken predominantly immediately post mortem and processed for transmission electron microscopy. As a result, electron micrographs of 90 horses in total were available for analysis comprising 4 control horses, 16 horses suffering from myopathy and 70 otherwise diseased horses. Results: Following a thorough clinical and laboratory work-up, four out of five patients that did not fit into the usual algorithm to detect known causes of myopathy showed ultrastructural mitochondrial alterations. Small mitochondria with zones with complete disruption of cristae associated with lactic acidemia were detected in a 17-year-old pony mare, extremely long and slender mitochondria with longitudinal cristae in a 5-year-old Quarter horse stallion, a mixture of irregular extremely large mitochondria (measuring 2500 by 800 nm) next to smaller ones in an 8-year-old Hanoverian mare and round mitochondria with only few cristae in a 11-year-old pony gelding. It remains uncertain whether the subsarcolemmal mitochondrial accumulations observed in the fifth patient have any pathological significance. Conclusions: Ultrastructural alterations in mitochondria were detected in at least four horses. To conclude that these are due to mitochondrial dysfuntions, biochemical tests should be performed. Practical applications: The possibility of a mitochondrial myopathy should be included in the differential diagnosis of muscle weakness.

Mycobacterium saopaulense sp. nov., a rapidly growing mycobacterium closely related to members of the Mycobacterium chelonae--Mycobacterium abscessus group

Five isolates of non-pigmented, rapidly growing mycobacteria were isolated from three patients and,in an earlier study, from zebrafish. Phenotypic and molecular tests confirmed that these isolates belong to the Mycobacterium chelonae-Mycobacterium abscessus group, but they could not be confidently assigned to any known species of this group. Phenotypic analysis and biochemical tests were not helpful for distinguishing these isolates from other members of the M. chelonae–M.abscessus group. The isolates presented higher drug resistance in comparison with other members of the group, showing susceptibility only to clarithromycin. The five isolates showed a unique PCR restriction analysis pattern of the hsp65 gene, 100 % similarity in 16S rRNA gene and hsp65 sequences and 1-2 nt differences in rpoB and internal transcribed spacer (ITS) sequences.Phylogenetic analysis of a concatenated dataset including 16S rRNA gene, hsp65, and rpoB sequences from type strains of more closely related species placed the five isolates together, as a distinct lineage from previously described species, suggesting a sister relationship to a group consisting of M. chelonae, Mycobacterium salmoniphilum, Mycobacterium franklinii and Mycobacterium immunogenum. DNA–DNA hybridization values .70 % confirmed that the five isolates belong to the same species, while values ,70 % between one of the isolates and the type strains of M. chelonae and M. abscessus confirmed that the isolates belong to a distinct species. The polyphasic characterization of these isolates, supported by DNA–DNA hybridization results,demonstrated that they share characteristics with M. chelonae–M. abscessus members, butconstitute a different species, for which the name Mycobacterium saopaulense sp. nov. is proposed. The type strain is EPM10906T (5CCUG 66554T5LMG 28586T5INCQS 0733T).

Early biochemical indicators of the obliterative bronchiolitis syndrome in lung transplantation

The diagnosis of the obliterative bronchiolitis syndrome in lung transplantation is presently best established by evaluation of postoperative lung function tests. Unfortunately the decline in lung function occurs only when obliteration has progressed significantly and is therefore not an early predictive indicator. To distinguish patients at increased risk for the development of obliterative bronchiolitis, we regularly assessed the chemiluminescence response of polymorphonuclear leukocytes, opsonic capacity, and plasma elastase/beta-N-acetylglucosaminidase in 52 outpatients (25 women and 27 men; mean age 45 +/- 12 years) who underwent transplantation between January 1991 and January 1992. Recent onset bronchiolitis within the described observation period occurred in 16 patients (group obliterative bronchiolitis). A matched cohort of 16 patients was formed according to type of procedure, age and follow-up (control) from the remaining 36 patients. Data obtained from a period 6 months before clinical onset of the syndrome showed a significant drop of the opsonic capacity (group obliterative bronchiolitis = 87% +/- 7%; control = 100% +/- 9%; p < 0.023) and rise of the N-acetyl-D-glucosaminidase (group obliterative bronchiolitis = 7.5 +/- 2 U/L; control = 5.8 +/- 1.8 U/L; p < 0.04). No correlation was found between the number of infectious events or rejection episodes and the incidence of obliterative bronchiolitis. According to these results, it can be concluded that a decrease in the plasma opsonic capacity and a rise in beta-N-acetylglucosaminidase may be early markers before clinical onset of obliterative bronchiolitis. The nonspecific immune system may therefore play an important role in the development of obliterative bronchiolitis.

Serum tests, liver stiffness and artificial neural networks for diagnosing cirrhosis and portal hypertension

BACKGROUND The diagnostic performance of biochemical scores and artificial neural network models for portal hypertension and cirrhosis is not well established. AIMS To assess diagnostic accuracy of six serum scores, artificial neural networks and liver stiffness measured by transient elastography, for diagnosing cirrhosis, clinically significant portal hypertension and oesophageal varices. METHODS 202 consecutive compensated patients requiring liver biopsy and hepatic venous pressure gradient measurement were included. Several serum tests (alone and combined into scores) and liver stiffness were measured. Artificial neural networks containing or not liver stiffness as input variable were also created. RESULTS The best non-invasive method for diagnosing cirrhosis, portal hypertension and oesophageal varices was liver stiffness (C-statistics=0.93, 0.94, and 0.90, respectively). Among serum tests/scores the best for diagnosing cirrhosis and portal hypertension and oesophageal varices were, respectively, Fibrosis-4, and Lok score. Artificial neural networks including liver stiffness had high diagnostic performance for cirrhosis, portal hypertension and oesophageal varices (accuracy>80%), but were not statistically superior to liver stiffness alone. CONCLUSIONS Liver stiffness was the best non-invasive method to assess the presence of cirrhosis, portal hypertension and oesophageal varices. The use of artificial neural networks integrating different non-invasive tests did not increase the diagnostic accuracy of liver stiffness alone.