19F electron nuclear double resonance (ENDOR) spectroscopy for distance measurements using trityl spin labels in DNA duplexes.

The combination of fluorine labeling and pulsed electron-nuclear double resonance (ENDOR) is emerging as a powerful technique for obtaining structural information about proteins and nucleic acids. In this work, we explored the capability of Mims 19F ENDOR experiments on reporting intermolecular distances in trityl- and 19F-labeled DNA duplexes at three electron paramagnetic resonance (EPR) frequencies (34, 94, and 263 GHz). For spin labeling, we used the hydrophobic Finland trityl radical and hydrophilic OX063 trityl radical. Fluorine labels were introduced into two positions of a DNA oligonucleotide. The results indicated that hyperfine splittings visible in the ENDOR spectra are consistent with the most populated interspin distances between 19F and the trityl radical predicted from molecular dynamic (MD) simulations. Moreover, for some cases, ENDOR spectral simulations based on MD results were able to reproduce the conformational distribution reflected in the experimental ENDOR line broadening. Additionally, MD simulations provided more detailed information about the melting of terminal base pairs of the oligonucleotides and about the configuration of the trityls relative to a DNA end.

CMAH genotyping survey for blood types A, B and C (AB) in purpose-bred cats.

In domestic cats, the AB blood group system consists of the three types A, B and C (also called AB). Mismatches can cause acute hemolytic transfusion reactions and hemolysis of the newborn (neonatal isoerythrolysis, NI). As blood types B and C are inherited recessively to A, breeders need to know the genotype to predict blood types in offspring and avoid NI. Several CMAH variants have been described as being associated with the b and ac alleles, and different genotyping schemes exist. Here, we genotyped 2145 cats with the original SNV panel, including SNVs c.142G>A and ∆-53, and our new scheme, with SNVs c.179G>T, c.268T>A and c.1322delT, to differentiate types A and B and added the SNV for the common ac (c.364C>T). Based upon the new scheme, all samples were assigned the correct genotype. No discordances appeared for the A allele, and new breed-specific SNVs (c.179G>T, c.1322delT) for the b allele were discovered. Furthermore, the genotypes A/ac (type A), ac /ac (C) and ac /b (C) could be detected. We found the variant c.179G>T in additional breeds: Ragdoll, Siberian, Scottish Fold, Chartreux, Neva Masquerade, British Shorthair and Highlander. Also, the variant c.364C>T was detected in additional breeds: Bengal, British Shorthair, Maine Coon, and Scottish Fold. We conclude that our new SNV panel is superior in genotyping cats than the original SNV panel and assures correct assignments of types A, B and C to assist veterinary clinicians and breeders to recognize, confirm and avoid blood incompatibilities such as acute hemolytic transfusion reactions and NI.

A novel MLPH variant in dogs with coat colour dilution.

Coat colour dilution may be the result of altered melanosome transport in melanocytes. Loss-of-function variants in the melanophilin gene (MLPH) cause a recessively inherited form of coat colour dilution in many mammalian and avian species including the dog. MLPH corresponds to the D locus in many domestic animals, and recessive alleles at this locus are frequently denoted with d. In this study, we investigated dilute coloured Chow Chows whose coat colour could not be explained by their genotype at the previously known MLPH:c.-22G>A variant. Whole genome sequencing of such a dilute Chow Chow revealed another variant in the MLPH gene: MLPH:c.705G>C. We propose to designate the corresponding mutant alleles at these two variants d1 and d2 . We performed an association study in a cohort of 15 dilute and 28 non-dilute Chow Chows. The dilute dogs were all either compound heterozygous d1 /d2 or homozygous d2 /d2 , whereas the non-dilute dogs carried at least one wildtype allele D. The d2 allele did not occur in 417 dogs from diverse other breeds. However, when we genotyped a Sloughi family, in which a dilute coloured puppy had been born out of non-dilute parents, we again observed perfect co-segregation of the newly discovered d2 allele with coat colour dilution. Finally, we identified a blue Thai Ridgeback with the d1 /d2 genotype. Thus, our data identify the MLPH:c.705G>C as a variant explaining a second canine dilution allele. Although relatively rare overall, this d2 allele is segregating in at least three dog breeds, Chow Chows, Sloughis and Thai Ridgebacks.

Two MC1R loss-of-function alleles in cream-coloured Australian Cattle Dogs and white Huskies.

Loss-of-function variants in the MC1R gene cause recessive red or yellow coat-colour phenotypes in many species. The canine MC1R:c.916C>T (p.Arg306Ter) variant is widespread and found in a homozygous state in many uniformly yellow- or red-coloured dogs. We investigated cream-coloured Australian Cattle Dogs whose coat colour could not be explained by this variant. A genome-wide association study with 10 cream and 123 red Australian Cattle Dogs confirmed that the cream locus indeed maps to MC1R. Whole-genome sequencing of cream dogs revealed a single nucleotide variant within the MITF binding site of the canine MC1R promoter. We propose to designate the mutant alleles at MC1R:c.916C>T as e1 and at the new promoter variant as e2 . Both alleles segregate in the Australian Cattle Dog breed. When we considered both alleles in combination, we observed perfect association between the MC1R genotypes and the cream coat colour phenotype in a cohort of 10 cases and 324 control dogs. Analysis of the MC1R transcript levels in an e1 /e2 compound heterozygous dog confirmed that the transcript levels of the e2 allele were markedly reduced with respect to the e1 allele. We further report another MC1R loss-of-function allele in Alaskan and Siberian Huskies caused by a 2-bp deletion in the coding sequence, MC1R:c.816_817delCT. We propose to term this allele e3 . Huskies that carry two copies of MC1R loss-of-function alleles have a white coat colour.

Bayesian optimization to estimate hyperfine couplings from 19F ENDOR spectra.

ENDOR spectroscopy is a fundamental method to detect nuclear spins in the vicinity of paramagnetic centers and their mutual hyperfine interaction. Recently, site-selective introduction of 19F as nuclear labels has been proposed as a tool for ENDOR-based distance determination in biomolecules, complementing pulsed dipolar spectroscopy in the range of angstrom to nanometer. Nevertheless, one main challenge of ENDOR still consists of its spectral analysis, which is aggravated by a large parameter space and broad resonances from hyperfine interactions. Additionally, at high EPR frequencies and fields (⩾94 GHz/3.4 Tesla), chemical shift anisotropy might contribute to broadening and asymmetry in the spectra. Here, we use two nitroxide-fluorine model systems to examine a statistical approach to finding the best parameter fit to experimental 263 GHz 19F ENDOR spectra. We propose Bayesian optimization for a rapid, global parameter search with little prior knowledge, followed by a refinement by more standard gradient-based fitting procedures. Indeed, the latter suffer from finding local rather than global minima of a suitably defined loss function. Using a new and accelerated simulation procedure, results for the semi-rigid nitroxide-fluorine two and three spin systems lead to physically reasonable solutions, if minima of similar loss can be distinguished by DFT predictions. The approach also delivers the stochastic error of the obtained parameter estimates. Future developments and perspectives are discussed.

NHLRC1 homozygous dodecamer expansion in a Newfoundland dog with Lafora disease.

Lafora disease is a genetic disease caused, in humans, by mutations in EPM2A and NHLRC1 genes, resulting in accumulation of polyglucosan bodies within neurons. Affected subjects present progressive neurological signs characterised primarily by myoclonic epilepsy. In dogs, Lafora disease has been described mainly in miniature wire-haired Dachshunds, where a dodecamer expansion in NHLRC1 gene has been identified. The same mutation has then been detected in the Basset Hound, Beagle, Chihuahua and Pembroke Welsh Corgi breeds. This is the first case of a Newfoundland dog with myoclonic epilepsy diagnosed with Lafora disease based on confirmed dodecamer expansion in the NHLRC1 gene. Lafora disease is being progressively recognised in different unrelated breeds suggesting a wider distribution in the canine population than previously thought.

Chromatofocusing combined with the ELISA technique. A sensitive method for the analysis of immune complexes.

A sensitive method which permits analysis of IgG containing circulating immune complexes without detailed knowledge of the nature of the antigens and the specificity of the antibodies involved is described. Soluble BSA: anti-BSA were used as model immune complexes and isolated from serum. The procedure involves the use of gel chromatography for the separation of the high molecular weight fraction containing the immune complexes as measured by binding to 125I-labeled Clq, followed by absorption of the immune complex fraction to immobilized protein A-Sepharose CL-4B. After desorption from protein A-Sepharose the complexes were dissociated and separated into free antigen and antibody by chromatofocusing in the presence of urea. The isolated free antigen and antibody retained their immunological activity as shown by immunodiffusion, binding after their recombination to 125I-labeled Clq, and by recombining antigen and antibody with much enhanced sensitivity using a microplate ELISA system. By means of the ELISA recombination technique it is possible to analyze less than 1 microgram of BSA:anti-BSA model complexes. Application of this technique may provide more information about the nature of immune complex like material associated with diseases.

Ultrasound-guided percutaneous fine needle aspiration biopsy of abdominal and retroperitoneal masses. Accuracy of cytology in the diagnosis of malignancy, cytologic tumor typing and use of antibodies to intermediate filaments in selected cases.

The reliability of ultrasound-guided fine needle aspiration biopsy (FNAB) in the detection of intraperitoneal and retroperitoneal malignancies was evaluated in 308 consecutive cases seen between 1979 and 1983. The prevalence of malignant neoplasms was 68.5%. The overall accuracy of FNAB diagnosis was 88.9%, with a sensitivity of 84.4% and a specificity of 98%. The predictive value of positive and negative results were 98.9% and 74.6%, respectively. Additionally, the cytologic results were statistically evaluated with respect to the different sites of the biopsied lesions (including pancreas, liver, kidneys and miscellaneous sites). The overall accuracy was highest for malignant lesions in the liver (96.4%) and in miscellaneous sites (89.5%). Reasons for false-negative results included incorrect areas sampled, limited material due to fibrosis or necrosis and cytologic misinterpretations. The accuracy of cytologic tumor typing with respect to histogenetic origin was 96.8%. Cytologic subclassification was performed with lower accuracy (82.5%), and exact determination of the site of the primary tumor from cytologic criteria alone was possible for 35.7% of carcinomas. In selected cases, routine cytologic examination was supplemented by intermediate filament typing using well-characterized antibodies against cytokeratin, vimentin, desmin and neurofilaments. Examples are shown in which use of this method clearly increased the accuracy of the diagnosis. Only two serious complications (bleeding) of fine-needle aspiration biopsy were encountered in this series.

G244E in the canine factor IX gene leads to severe haemophilia B in Rhodesian Ridgebacks.

Haemophilia B in Rhodesian Ridgebacks is currently the most important canine haemophilia in Germany. The aim of this study was to define the underlying genetic defect. Genetic studies were performed including six phenotypically affected male dogs (factor IX activity: approximately 1%), four suspected carriers (factor IX activity 48-69%, one confirmed by affected offspring), and 12 healthy dogs. Comparison of the entire coding region of the canine factor IX DNA sequences and exon-intron junctions from affected dogs with the wild type canine factor IX DNA revealed a G-A missense mutation in exon 7. This mutation results in a glycine (GGA) to glutamic acid (GAA) exchange in the catalytic domain of the haemophilic factor IX. All affected dogs were hemizygous for the detected mutation and carriers were heterozygous, whereas none of the Rhodesian Ridgebacks with normal factor IX activity showed the mutation. No further alterations in the sequences between affected dogs and the healthy control group could be observed. None of the Rhodesian Ridgebacks with undefined haemophilia B status (n=30) and no individual of three other dog breeds (Doberman Pinscher: n=20; German Wire haired Pointer: n=20; Labrador: n=25) showed the presence of the mutation. Amino acid sequence alignment and protein structural modelling analysis indicate that the detected mutation causes a relevant functional defect. The results of this study suggest that the detected mutation is responsible for a severe form of haemophilia B in Rhodesian Ridgebacks.

[Right bundle-branch block configuration in the pacemaker ECG--3 different observations]. / Rechtsschenkelblock-Konfiguration im Schrittmacher-EKG--3 unterschiedliche Beobachtungen.

We report on 3 cases of right bundle branch block configuration in the ECG after pacemaker implantation. In case 1 it was not noticed that the catheter had been directed through an unknown sinus venosus defect into the left atrium and the left ventricle. In case 2 the catheter reached the left ventricle via an iatrogenic perforation of the lower part of the atrial septum. In case 3 the catheter was situated correctly in the right ventricle, but the tip was penetrating the septum. Different possibilities of interpretation of this phenomenon are discussed, and a description of further pacemaker catheter misplacements which may be responsible for a right bundle branch block configuration is given. The authors take the view that a pacemaker catheter should not remain in the arterial area, in the pericardial space and in the coronary sinus. A lateral radiograph, a 12-lead ECG and an echocardiographic study are helpful for detecting and interpreting an unusual catheter position or a right bundle branch block configuration in the pacemaker ECG.

Solid-phase radioimmunoassay for detection of alcoholic hyalin antigen (AHAg) and antibody (anti-AH).

Alcoholic hyalin (AH) was isolated and purified from post-mortem livers of patients with alcoholic hepatitis. Antibodies against AH (anti-AH) were raised in guinea-pigs. Their specificity was demonstrated by immunofluorescence and by immunoabsorption. The antisera were positive in immunofluorescence and complement fixation up to serum dilutions of 1:320 and 1:32 respectively. In order to achieve a higher sensitivity a solid-phase radioimmunoassay (SP-RIA) was established for detection of alcoholic hyalin antigen (AHAg) and anti-AH. Anti-AH could thus be measured up to a 10(5) serum dilution. Using a blocking test, solubilized AH at protein concentrations as low as 80 to 160 ng/ml could be detected. With this SP-RIA, neither AHAg nor anti-AH was found in the sera of 32 patients with histologically proven alcoholic hepatitis. The sera were likewise negative by indirect immunofluorescence, complement fixation and immune adherence haemagglutination test.

Does an antifoaming agent improve the quality of abdominal ultrasonography? A double-blind study with special reference to the pancreas, the aorta and the para-aortic region.

To overcome the effects of intestinal gas which may be a problem for ultrasound examinations, a double-blind study was undertaken in 42 patients in whom the first ultrasound examination was unsatisfactory due to excessive intestinal gas. The patients received either a liquid dimeticon preparation over 24 hours (total dosage 480 mg) or a placebo. Criteria for the evaluation of the treatment were the ability to visualize the pancreas, the aorta and the para-aortic region at the second examination. The quality of the ultrasonic images was found to be improved in 52% of the controls by repetition and in 71% of the test substance group, the difference being not significant (p greater than 0.05). It is concluded that: It is worthwhile asking the patient to return for a second examination 24 hours later before resorting to invasive and/or expensive procedures when the first scan was of poor quality and liquid dimeticon has no significant defoaming effect in these patients.

Overwhelming infection after splenectomy in spite of some spleen remaining and splenosis. A case report.

A fatal case of overwhelming postsplenectomy pneumococcal sepsis is presented occurring in a 37-year-old female 11 years after removal of the spleen because of traumatic rupture. The patient died 11 h after admission to hospital and about 32 h after sudden onset of illness. At necropsy splenic tissue, splenosis, disseminated intravascular coagulation, and thrombi within the arterioles consisting of gram-positive cocci and adrenal hemorrhage were found. The clinical, laboratory, and postmortem findings are described. Reports had been published of 41 other cases of overwhelming postsplenectomy infection (OPSI) in patients aged 20 years or more, but only three of these cases of OPSI syndrome occurred in spite of remaining splenic tissue. The longest interval between extirpation of spleen and subsequent sepsis was 42 years, indicating a small but lifelong risk of severe infection in asplenic patients. In view of the literature, the role of spleen in infection defence, the splenic function in blood clearance, and the prevention of postsplenectomy infections by antibiotic prophylaxis, pneumococcal vaccine, and reimplantation of autochthonous splenic tissue or infrared contact coagulation are discussed.

Critical aspects of the 125I-C1q binding assay for detecting immune complexes in tumor patients.

Clinical studies on C1q binding activity in tumor patients showed contradictory results and it was suggested that methodical problems might be partly responsible for those discrepancies. Therefore, precision of the C1q binding assay when testing tumor sera, influences of assay modifications on test results and possible interfering substances were investigated. Compared to aggregated human IgG and BSA: anti-BSA complexes as standards the C1q binding material in tumor sera was more unstable after freezing-thawing and distinctly more susceptible to methodical influences like testing with different 125I-C1q preparations or variations of PEG concentration and ionic strength of PEG solution. Addition of heparin and fibrinogen influenced the C1q binding of some tumor sera more than the C1q binding of standards and normal sera. Thus, instability and sensitivity to methodical influences of the C1q binding material have to be considered, when clinical studies using the C1q binding assay to detect immune complexes in tumor sera are interpreted.

Immune complex-like material in the serum and plasma exchange in patients with metastatic cancer.

Clinical significance of immune complex-like material in the serum was investigated in tumour patients undergoing plasma exchange with albumin-saline solution and subsequent chemotherapy. Immune complexes were detected by the Clq binding assay or the Raji cell radioimmunoassay in nine out of forty-five patients before this therapy. Levels of immune complexes were decreased to 10-30% of the initial value by plasma exchange depending on exchanged plasma volume. In contrast to other serum proteins like alpha 1-antitrypsin and alpha 2-macroglobulin, which showed protein specific increase during follow up after plasma exchange in all patients, recovery rates of immune complexes and IgG were highly individual but parallel in each patient. Clinical response to this protocol did not correlate with immune complex status, suggesting that removal of the measured immune complex like material had little clinical significance or was not longlasting enough to provide therapeutic benefit.

[Clinical significance of circulating immune complexes in patients with metastatic breast cancer]. / Klinische Wertigkeit der Bestimmung zirkulierender Immunkomplexe bei Patientinnen mit metastasierendem Mammakarzinom.

In 68 patients with metastatic breast cancer a follow-up study was performed to correlate circulating immune complexes (CIC) as detected by the C1q binding assay and the Raji cell radio immunoassay with the state of disease. Clinical examinations and determinations of CIC were carried out all four to eight weeks over at least six months. 19 patients were positive for CIC in the C1q binding assay and 12 in the Raji cell radioimmunoassay. There was no correlation between the results of both tests. In comparison 26 patients out of 68 with rheumatoid arthritis were positive in the C1q binding assay and 32 in the Raji cell assay. In these patients the results of both tests correlated significantly. There was only in a few cases of metastatic breast cancer a positive correlation between levels of CIC and changes of tumor burden. Furthermore, CIC did not prove to be of prognostic value.

Clinical significance of circulating immune complexes in patients with metastatic breast cancer.

Serum circulating immune complexes (CIC) were repeatedly measured by means of the CIq binding assay (Cba) and the Raji cell assay (Rca) in 158 patients with metastatic breast cancer (mbc). Frequency of occurrence and levels of CIC were only slightly increased in mbc when compared to age-matched healthy women. They were identical to those of patients with localized breast cancer prior to mastectomy and to those of post mastectomy patients without evidence of recurrent disease. In mbc the results of the Cba and the Rca showed a poor correlation, whereas in a control group of patients with rheumatoid arthritis both tests showed significantly elevated levels of CIC. Patients with mbc were followed up clinically and biochemically by serially measuring CIC for an average of 10 months. Patients with positive CIC did not prove to be an unfavorable group regarding progression of disease and response to chemotherapy. When CEA and CIC levels were compared, CIC, unlike CEA, was a poor tumor marker. In conclusion, CIC as measured by CIq binding assay and Raji cell assay are not clinically significant tumor markers or prognostic indicators in patients with metastatic breast cancer.

[First clinical experience with large volume plasma exchange in malignant tumor patients]. / Erste klinische Erfahrungen mit dem grossvolumigen Plasmaaustausch bei Patienten mit malignen Erkrankungen.

An attempt has been made to standardize the method of large volume plasma exchange in tumor patients on the basis of an albumin-electrolyte exchange solution. Problems relating to the alteration of tumor patients' serum proteins are discussed. Three patients with malignancies successfully underwent therapeutic plasmaphereses.