Generation of a human iPSC line (HIMRi001-A) from a patient with filaminopathy.

Here we introduce the human induced pluripotent stem cell (hiPSC) line HIMRi001-A generated from cultured dermal fibroblasts of a 60-year-old male patient with a myofibrillar myopathy, carrying a heterozygous c.4984C > T [p.Q1662X] mutation in the filamin C (FLNC)-gene, via lentiviral expression of OCT4, SOX2, KLF4 and c-MYC. HIMRi001-A displays typical embryonic stem cell-like morphology, carries the c.4984C > T FLNC gene mutation, expressed several pluripotent stem cell makers, retained normal karyotype (46, XY) and holds the potential to differentiate in all three germ layers. We postulate that HIMRi001-A can be used for the elucidation of FLNC-associated pathomechanisms and for developing new therapeutic options.

Pulse transit time and blood pressure during cardiopulmonary exercise tests.

Pulse transit time (PTT), the interval between ventricular electrical activity and peripheral pulse wave, is assumed to be a surrogate marker for blood pressure (BP) changes. The objective of this study was to analyze PTT and its relation to BP during cardiopulmonary exercise tests (CPET). In 20 patients (mean age 51+/-18.4 years), ECG and finger-photoplethysmography were continuously recorded during routine CPETs. PTT was calculated for each R-wave in the ECG and the steepest slope of the corresponding upstroke in the plethysmogram. For each subject, linear and non-linear regression models were used to assess the relation between PTT and upper-arm oscillometric BP in 9 predefined measuring points including measurements at rest, during exercise and during recovery. Mean systolic BP (sBP) and PTT at rest were 128 mm Hg and 366 ms respectively, 197 mm Hg and 289 ms under maximum exercise, and 128 mm Hg and 371 ms during recovery. Linear regression showed a significant, strong negative correlation between PTT and sBP. The correlation between PTT and diastolic BP was rather weak. Bland-Altman plots of sBP values estimated by the regression functions revealed slightly better limits of agreements for the non-linear model (-10.9 to 10.9 mm Hg) than for the linear model (-13.2 to 13.1 mm Hg). These results indicate that PTT is a good potential surrogate measure for sBP during exercise and could easily be implemented in CPET as an additional parameter of cardiovascular reactivity. A non-linear approach might be more effective in estimating BP than linear regression.

High-sensitivity fluorescence anisotropy detection of protein-folding events: application to alpha-lactalbumin.

An experimental procedure has been devised to record simultaneously fluorescence intensity and fluorescence anisotropy. A photoelastic modulator on the excitation beam enables the anisotropy signal to be recorded in one pass using a single photomultiplier tube and eliminates the need for a polarizer on the emission path. In conjunction with a stopped-flow mixer, providing a time-resolved capability, this procedure was used to study the refolding of apo alpha-lactalbumin following dilution from guanidinium chloride. Although the fluorescence intensity does not change detectably, the fluorescence anisotropy was found to resolve the conformational changes occurring between the initial unfolded state and the molten globule state formed either kinetically during refolding at pH 7.0 or at equilibrium at pH 2.0 (A-state). This result provides further evidence that fluorescence anisotropy is a valuable probe of protein structural transitions and that the information it provides concerning the rotational mobility of a fluorophore can be complementary to the information about the local environment provided by fluorescence intensity.

EGF-like domain calcium affinity modulated by N-terminal domain linkage in human fibrillin-1.

Calcium binding epidermal growth factor-like domains (cbEGFs) are present in many extracellular proteins, including fibrillin-1, Notch-3, protein S, factor IX and the low density lipoprotein (LDL) receptor, which perform a diverse range of functions. Genetic mutations that cause amino acid changes within these proteins have been linked to the Marfan syndrome (MFS), CADASIL, protein S deficiency, haemophilia B and familial hypercholesterolaemia, respectively. A number of these mutations disrupt calcium binding to cbEGFs, emphasising the critical functional role of calcium in these proteins. We have determined the calcium binding affinity of two sites within a cbEGF pair (cbEGF12-13) from human fibrillin-1 using two-dimensional nuclear magnetic resonance (NMR) and fluorescence techniques. Fibrillin-1 is a mosaic protein containing 43 cbEGF domains, mainly arranged as tandem repeats. Our results show that the cbEGF13 site in the cbEGF12-13 pair possesses the highest calcium affinity of any cbEGF investigated from fibrillin-1. A comparative analysis of these and previously reported calcium binding data from fibrillin-1 demonstrate that the affinity of cbEGF13 is enhanced more than 70-fold by the linkage of an N-terminal cbEGF domain. In contrast, comparison of calcium binding by cbEGF32 in isolation relative to when linked to a transforming growth factor beta-binding protein-like domain (TB6-cbEGF32) reveals that the same enhancement is not observed for this heterologous domain pair. Taken together, these results indicate that fibrillin-1 cbEGF Ca2+ affinity can be significantly modulated by the type of domain which is linked to its N terminus. The cbEGF12-13 pair is located within the longest contiguous section of cbEGFs in fibrillin-1, and a number of mutations in this region are associated with the most severe neonatal form of MFS. The affinities of cbEGF domains 13 and 14 in this region are substantially higher than in the C-terminal region of fibrillin-1. This increased affinity may be important for fibrillin assembly into 10-12 nm connective tissue microfibrils and/or may contribute to the biomechanical properties of the microfibrillar network.

Evaluation of SICKLEQUIK, a differential solubility test for hemoglobin S.

Researchers evaluated a differential tube solubility test's ability to detect Hemoglobin S (Hb S) in sickle cell conditions. Six hundred twenty-nine blood samples, including 190 S hemoglobinopathies, were used to assess test performance. Alkaline cellulose acetate electrophoresis was used to confirm all results. The test correctly indicated the presence of Hb S in every case in which Hb S was found on electrophoresis. Six of the 198 specimens containing Hb S incorrectly differentiated the heterozygous from the homozygous state (two with Hb AS, three with Hb SF, and one with Hb SC). Electrophoresis later showed four of these samples to be inappropriate for solubility testing. Three of these specimens contained Hb SF and the other specimen contained Hb SC. One specimen from a patient with polycythemia gave a false positive result. Results were compared using different methods of centrifugation. This solubility test was found to be dependable as a screening method provided the manufacturer's directions are followed and all positive results are confirmed by electrophoresis.

[Familial hemophagocytic reticulosis (Farquhar) (author's transl)]. / Familiäre hämophagozytierende Retikulose (Farquhar).

An account is given of six cases of familial hemophagocytic reticulosis (familial lymphohistiocytosis). After a period of illness varying from 2 weeks to 7.5 months the infants studied died with pancytopenia, fever and hepatosplenomegaly. Histologic examination revealed three characteristic features: Lymphocyte infiltration of the organs, reduction of the lymphatic and hematopoetic system, histiocytosis with erythrophagocytosis. The etiology of this disease remains obscure. Congenital allogeneity may be a possibility, but further immunologic investigations would be necessary to support this hypothesis.

"Fast isoamylases" in the parotid saliva of children with cystic fibrosis and heterozygous carriers.

On electrophoresis, parotid saliva always exhibits a basic pattern of 6 isoamylases. Additional faster migrating isoamylases occur in varying numbers. These "fast isoamylases" are generated, at least in part, by deamidation. Compared with juvenile and adult controls, a significantly greater number of "fast isoamylases" was found in the parotid saliva of children with cystic fibrosis and their healthy heterozygous parents. A shift in the equilibrium between amidation and deamidation is discussed in terms of its possible connection with the metabolic defect responsible for cystic fibrosis.

[Fast isoamylases in saliva from mucoviscidosis patients (author's transl)]. / Uber das Auftreten schneller Isoamylasen im Mukoviszidosespeichel

Human parotid saliva is characterized by a basic pattern of 6 isoamylases; during ageing, it contains additional isoamylases which migrate faster towards the anode. An increasing number of 'fast isoamylases' are also found in saliva from mucoviscidosis patients. Their possible symptomatological significance for the pathogenesis of mucoviscidosis which is as yet not solved is being discussed.

'Fast isoamylases' in parotid saliva of heterozygous carriers of cystic fibrosis.

An increased number of 'fast isoamylases' is found in the parotid saliva of children with cystic fibrosis and their heterozygous parents who are clinically healthy. 'Fast isoamylases' are generated, at least in part, from the 6 isoamylases of the basic pattern by deamidation of asparagine and glutamine residues. This basic pattern is present in the parotid saliva of all subjects we have tested so far. A shift in the equilibrium between amidation and deamidation processes is discussed in terms of its possible significance for the pathogenesis of cystic fibrosis and for ageing.