Early diagnosis in Duchenne muscular dystrophy.
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Child, Preschool↗
Biomedical subjects
K Wagner
Publications and source records attributed to K Wagner.
Mosaicism in a fragile X male including a de novo deletion in the FMR1 gene.
In most cases the fragile X syndrome is caused by an amplification of the CGG trinucleotide repeat in the 5' untranslated region of the FMR1 gene, in combination with the hypermethylation of the proximal CpG island. Recently, also a few cases with deletions or a mosaic of a deletion and a full mutation in the FMR1 gene, leading to the same phenotype, have been described. Here we report the molecular analysis of a patient with typical fragile X phenotype and mosaicism of the FMR1 genomic region consisting of a premutation, a full mutation of the CGG repeats, and a 215 bp deletion, diagnosed by Southern blot hybridisation and polymerase chain reaction (PCR). Sequence analysis of the deletion demonstrated that the 5' breakpoint of the deletion is located within a putative hotspot region 75-53 bp proximal to the CGG repeat.
Adult↗
Cloning and characterization of the transport modifier RS1 from rabbit which was previously assumed to be specific for Na+-D-glucose cotransport.
Previously we cloned membrane associated polypeptides from pig and man (pRS1, hRS1) which altered rate and glucose dependence of Na+-d-glucose cotransport expressed by SGLT1 from rabbit and man. This paper describes the cloning of a related cDNA sequence from rabbit intestine (rbRS1) which encodes a gene product with about 65% amino acid identity to pRS1 and hRS1. Hybridization of endonuclease-restricted genomic DNA with cDNA fragments of rbRS1 showed that there is only one gene with similarity to rbRS1 in rabbit, and genomic PCR amplifications revealed that the rbRS1 gene is intronless. Comparing the transcription of rbRS1 and rbSGLT1 in various tissues and cell types, different mRNA patterns were obtained for both genes. In Xenopus oocytes the Vmax of expressed Na+-d-glucose cotransport was increased or decreased when rbRS1 was coexpressed with rbSGLT1 or hSGLT1, respectively. After coexpression with hSGLT1 the glucose dependence of the expressed transport was changed. By coexpression of rbRS1 with the human organic cation transporter hOCT2 the expressed cation uptake was not altered; however, the expressed cation uptake was drastically decreased when hRS1 was coexpressed with hOCT2. The data show that RS1 can modulate the function of transporters with non-homologous primary structures.
Amino Acid Sequence↗
Enhancement of human IL-4 activity by soluble IL-4 receptors in vitro.
The recombinant form of the extracellular domain of the IL-4 receptor (sIL-4R) is a potential candidate to neutralize IL-4; however, murine sIL-4R displayed both antagonistic and agonistic activity in vivo. Here we show that human recombinant sIL-4R induced the formation of complexed IL-4 in supernatants of activated T cells in a dose-dependent manner as measured by newly developed enzyme-linked immunosorbent assays. These IL-4/sIL-4R complexes liberated free IL-4 even after prolonged culturing. In contrast, in the absence of exogenously added sIL-4R, free IL-4 was rapidly consumed or proteolytically degraded in cultures of activated T cells. Thus, no IL-4 bioactivity could be determined in supernatants of T cells activated in the presence of IL-4 for 6 days. In contrast, the same cultures carried out in the presence of sIL-4R showed marked IL-4 bioactivity. While low concentrations of sIL-4R enhanced IL-4-driven inhibiton of IFN-gamma production by activated T cells, higher concentrations neutralized IL-4. Together, human sIL-4R, besides its activity as an antagonist to IL-4, also possesses protective and agonistic functions for IL-4, which may be relevant for clinical studies aiming to neutralize IL-4 in vivo.
Animals↗
Safe biotechnology 9: values in risk assessment for the environmental application of microorganisms. The Safety in Biotechnology Working Party of the European Federation of Biotechnology.
Risk assessment for the deliberate release of microorganisms into the environment is traditionally carried out on a case-by-case basis. In a similar approach to that used when assessing human pathogenicity, we propose an alternative approach by introducing risk classes to facilitate or complement this type of risk assessment. These consider several sets of scenarios that address the different values that need to be protected. Examples of this approach include risk-class definitions for soil fertility and biodiversity.
Bacteria↗
Assignment of STAC to human chromosome band 3p22.3 between D3S3718 and D3S1611.
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Chromosome Banding↗
Urokinase activates the Jak/Stat signal transduction pathway in human vascular endothelial cells.
Endothelial cells demonstrate high urokinase expression and upregulation of urokinase receptors in response to vascular injury. Urokinase receptor binding facilitates endothelial cell migration into an arterial wound; however, the signaling cascade induced by the urokinase receptor in this cell type is incompletely understood. Because the Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway seems to be important for vessel function, we investigated the hypothesis that urokinase receptor binding activates Jak/Stat signaling in human vascular endothelial cells. Incubation of endothelial cells with urokinase-type plasminogen activator (uPA,1 nmol/L) induced a rapid and pronounced increase in tyrosine phosphorylation of several proteins with a molecular weight between 80 to 90 and 130 to 140 kDa. The same pattern of tyrosine phosphorylation was found after treatment with 1 nmol/L ATF, the urokinase amino-terminal fragment, which is devoid of proteolytic activity but still binds to the urokinase receptor. Using coimmunoprecipitation techniques, we demonstrated that the activated urokinase receptor is associated with 2 cytoplasmic tyrosine kinases of the Jak family, viz, Jak1 and Tyk2. uPA and ATF induced a time-dependent activation of both kinases, as shown by immunoprecipitation and Western blot analysis. Using electrophoretic mobility shift and supershift assays, we then demonstrated that Stat1 is rapidly activated in endothelial cells in response to uPA and ATF. Furthermore, Stat1 specifically binds to the regulatory elements interferon-gamma activation site/interferon-stimulated response element. The uPA-induced, time-dependent translocation of Stat1 to cell nuclei was confirmed by confocal microscopy study and immunoblotting of nuclear extracts with an anti-Stat1 antibody. This study provides evidence for a novel signaling pathway for uPA in human vascular endothelial cells. Direct activation of the Jak/Stat system via the uPA-receptor complex may be an important mechanism for endothelial cell migration and/or proliferation during angiogenesis and after vascular injury.
Biological Transport↗
[Screening for thrombophilia].
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Blood Coagulation Tests↗
Detection of and discrimination between total and free human interleukin-4 and free soluble interleukin-4 receptor by ELISA.
Interleukin-4 (IL-4) signaling is initiated by binding of IL-4 to the high-affinity IL-4 receptor alpha-chain and subsequent interaction with the common gamma-chain. Soluble forms of the extracellular domain of the alpha-chain (sIL-4R) were shown to be present in biological fluids and, dependent on the concentration, enhance or inhibit IL-4 activity by forming IL-4/sIL-4R complexes. To discriminate between free and potentially active IL-4 from the inactive and complexed form, we have established a set of new ELISA systems for the measurement of human IL-4 in its distinct forms. To select suitable pairs of anti-IL-4 antibodies, a chequerboard interference analysis with six highly-selective human IL-4 specific monoclonal antibodies was performed. For the determination of total IL-4, a monoclonal capture antibody was used that binds IL-4 outside the binding site of the IL-4R alpha-chain. Another antibody recognizing an epitope of the alpha-chain binding site was chosen for the detection of free IL-4. The binding of this antibody was inhibited in a dose-dependent fashion by recombinant sIL-4R. Assays for both total and free IL-4 exhibited a sensitivity of 8 pg/ml and a dynamic range up to 1000 pg/ml. Human sIL-4R was detected by two monoclonal antibodies directed against different epitopes. This ELISA was inhibited by recombinant IL-4 suggesting the measurement of predominantly free sIL-4R. Complexes between soluble IL-4R and IL-4 were detected by a monoclonal anti-sIL-4R antibody in combination with an anti-IL-4 antibody. When supernatants of activated T cells were analyzed, the majority of the IL-4 was in free form. The amount of complexed IL-4 was low as indicated by the fact that most of total IL-4 could be detected as free IL-4. Although values obtained for complexed IL-4 correlated with the difference between total and free IL-4, precise values could not be determined, presumably due to the dynamic nature of the complex between the two proteins. We suggest that the ability to quantitate total and free IL-4 in combination with sIL-4R may provide a new insight of the role that IL-4 plays in different pathophysiological conditions.
Antibodies, Monoclonal↗
Transport of the new chemotherapeutic agent beta-D-glucosylisophosphoramide mustard (D-19575) into tumor cells is mediated by the Na+-D-glucose cotransporter SAAT1.
For beta-D-glucosylisophosphoramide mustard (beta-D-Glc-IPM), a new alkylating drug in which isophosphoramide mustard is stabilized, a higher selectivity and lower myelotoxicity was observed than for the currently used cytostatic ifosfamide. Because beta-D-Glc-IPM is hydrophilic and does not diffuse passively through the lipid bilayer, we investigated whether a transporter may be involved in the cellular uptake. A variety of cloned Na+-sugar cotransporters were expressed in Xenopus oocytes, and uptake measurements were performed. By tracer uptake and electrical measurements it was found that beta-D-Glc-IPM was transported by the low-affinity Na+-D-glucose cotransporter SAAT1, which had been cloned from pig and is also expressed in humans. At membrane potentials between -50 and -150 mV, a 10-fold higher substrate affinity (Km approximately 0.25 mM) and a 10-fold lower Vmax value were estimated for beta-D-Glc-IPM transport than for the transport of D-glucose or methyl-alpha-D-glucopyranoside (AMG). Transport of beta-D-Glc-IPM and glucose by SAAT1 is apparently performed by the same mechanism because similar sodium dependence, dependence on membrane potential, electrogenicity, and phlorizin inhibition were determined for beta-D-Glc-IPM, D-glucose, and AMG. Transcription of human SAAT1 was demonstrated in various human carcinomas and tumor cell lines. In one of these, the human carcinoma cell line T84, phlorizin inhibitable uptake of beta-D-Glc-IPM was demonstrated with substrate saturation and an apparent Km of 0.4 mM. The data suggest that the Na+-D-glucose cotransporter SAAT1 transports beta-D-Glc-IPM into human tumor cells and may accumulate the drug in the cells. They provide an example for drug targeting by employing a plasma membrane transporter.
Animals↗
Characterization of human type X procollagen and its NC-1 domain expressed as recombinant proteins in HEK293 cells.
Type X collagen is a short-chain, network-forming collagen found in hypertrophic cartilage in the growth zones of long bones, vertebrae, and ribs. To obtain information about the structure and assembly of mammalian type X collagen, we generated recombinant human type collagen X by stable expression of full-length human alpha1(X) cDNA in the human embryonal kidney cell line HEK293 and the fibrosarcoma cell line HT1080. Stable clones were obtained secreting recombinant human type X collagen (hrColX) in amounts of 50 microg/ml with alpha1(X)-chains of apparent molecular mass of 75 kDa. Pepsin digestion converted the native protein to a molecule migrating as one band at 65 kDa, while bands of 55 and 43 kDa were generated by trypsin digestion. Polyclonal antibodies prepared against purified hrColX reacted specifically with type X collagen in sections of human fetal growth cartilage. Circular dichroism spectra and trypsin/chymotrypsin digestion experiments of hrColX at increasing temperatures indicated triple helical molecules with a reduced melting temperature of 31 degrees C as a result of partial underhydroxylation. Ultrastructural analysis of hrColX by rotary shadowing demonstrated rodlike molecules with a length of 130 nm, assembling into aggregates via the globular noncollagenous (NC)-1 domains as reported for chick type X collagen. NC-1 domains generated by collagenase digestion of hrColX migrated as multimers of apparent mass of 40 kDa on SDS-polyacrylamide gel electrophoresis, even after reduction and heat denaturation, and gave rise to monomers of 18-20 kDa after treatment with trichloroacetic acid. The NC-1 domains prepared by collagenase digestion comigrated with NC-1 domains prepared as recombinant protein in HEK293 cells, both in the multimeric and monomeric form. These studies demonstrate the potential of the pCMVsis expression system to produce recombinant triple helical type X collagens in amounts sufficient for further studies on its structural and functional domains.
Cell Line↗
Roussy-Lévy syndrome is a phenotypic variant of Charcot-Marie-Tooth syndrome IA associated with a duplication on chromosome 17p11.2.
The Roussy-Lévy syndrome (MIM #180800) was described in 1926 as a disorder presenting with pes cavus and tendon areflexia, distal limb weakness, tremor in the upper limbs, gait ataxia and distal sensory loss. We report a family with affected members in four generations, showing these clinical signs of Roussy-Lévy syndrome and a partial duplication at chromosome 17p11.2. This genetic defect is commonly found in patients with the hypertrophic form of the Charcot-Marie-Tooth syndrome. Our finding provides evidence against the Roussy-Lévy syndrome as a distinct entity but suggests a close relation with the Charcot-Marie-Tooth syndrome. What causes the additional features of gait ataxia and essential tremor needs further clarification.
Action Potentials↗
[Radiotherapy in surgical and nonsurgical patients. Therapy expectations, quality of life and physician assessment].
The present study investigates patients' expectations toward radiotherapy and their associations to quality of life and physician judgements. Fifty-five patients with tumors of different sites (30 with previous tumor-related surgery, 25 without surgery) admitted to the department of radiotherapy filled out a standardized questionnaire (EORTC QLQ-C30, PLC by Siegrist et al., therapy-related expectations and success) before and after inpatient radiotherapy. The corresponding physician ratings were collected. Fifty-eight percent of the patients expected the therapeutic goal "healing", whereas from the physician's standpoint this was realistic in only 7% of cases. The specific radiotherapy-related expectations "tumor control" and "pain relief" reached almost the same levels in patients and physician (71% vs 71% and 40% vs 44%). Patients with healing expectancy reported higher quality of life at the beginning of the therapy (53.4% vs 39.9%); patients expecting pain relief reported lower quality of life (37.1% vs 54.5%). Surgical patients who had been operated on within the past year (n = 18) showed a particularly high healing expectancy (83%), whereas patients whose operation dated back more than 1 year focused on pain relief as therapeutic goal (83%). The surgeon, as the primary contact person for patients, can influence patients' therapy-related expectations. In explaining the overall therapeutic strategy, surgeons should also mention the scope and limits of adjuvant therapies.
Adult↗
Donor-derived Plasmodium vivax infection following volunteer unrelated bone marrow transplantation.
A 14-year-old girl from the UK underwent volunteer unrelated donor bone marrow transplant (VUD BMT) for accelerated phase chronic myeloid leukaemia. On day +40 she became febrile, and peripheral blood smears demonstrated a 1% Plasmodium vivax parasitaemia. Although she had never been outside the UK, her male donor had documented Plasmodium vivax infection during a vacation in Papua New Guinea. Following appropriate treatment, he had been asymptomatic for 11 months before marrow harvesting. This is the first case report of malarial transmission by VUD BMT, and illustrates the potential problem of recrudescence of latent, dormant forms of Plasmodium vivax infection following transplantation into an immuno-compromised recipient. Even after appropriate therapy, malarial infection should be included in the differential diagnosis for all post-transplant febrile episodes.
Adolescent↗
Perioperative cytokine release during coronary artery bypass grafting in patients of different ages.
Surgical interventions and cardiopulmonary bypass (CPB) induce a systemic inflammatory response with cytokine release. Ageing is perceived as a process of impaired immune functions: IL-1beta, IL-6 and tumour necrosis factor-alpha (TNF-alpha) secretion are increased while IL-2 release is reduced in advanced age. At present, little information is available about perioperative immune reactions at different stages of ageing. The aim of the present study was to compare IL-6, IL-1beta, TNF-alpha, IL-10 and soluble IL-2 receptor (sIL-2R) in younger and older patients undergoing cardiac surgery. Male patients (n = 14) undergoing elective coronary artery bypass grafting (CABG) surgery employing CPB with moderate hypothermia were divided into two groups according to their age: group 1 included seven patients < 50 years old, group 2 included seven patients > 65 years old. All patients received general anaesthesia using a balanced technique with sufentanil, isoflurane and midazolam. Blood samples were collected pre-operatively (T1); intra-operatively during CPB (T2); post-operatively on the day of surgery (T3); on the first post-operative day (T4). Blood concentrations of IL-6, IL-1beta, IL-10, TNF-alpha and sIL-2R were measured using commercially available ELISA kits and corrected for plasma cell volume. Statistical analysis was performed by non-parametric analysis of variance and Mann-Whitney U-test. Significance level was set to P<0.05. There were no statistically significant differences in the perioperative release of TNF-alpha, IL-6, IL-1beta, IL-10 and sIL-2R among the two groups. We conclude that the perioperative course of cytokine release in patients undergoing CABG surgery with CPB and comparable perioperative management does not significantly differ in the two age groups.
Adult↗
Sublingual midazolam premedication in children: a dose response study.
The purpose of this study was to evaluate various doses of sublingual midazolam premedication in children. In our prospective, double-blind, placebo-controlled trial, children (n = 102, age range 12 to 129 months) scheduled for day surgery were randomized to receive either midazolam in one of three doses (0.25, 0.5, or 0.75 mg.kg-1) or placebo. Injectable midazolam was mixed with a thick grape syrup and placed under the tongue; the patient was asked to hold it as long as possible before swallowing. Children readily accepted the mixture. Analysing all patients randomized, none of the children receiving placebo vs 28% receiving 0.25 mg.kg-1 (P = 0.02), 52% receiving 0.5 mg.kg-1 (P < 0.001), and 64% receiving 0.75 mg.kg-1 (P < 0.001) of midazolam showed satisfactory sedation (drowsy) at 15 min after administration. Children receiving the two higher doses of midazolam (0.5 and 0.75 mg.kg-1) accepted mask induction willingly, while the group receiving 0.25 mg.kg-1 resembled the placebo group (P < 0.05).
Adjuvants, Anesthesia↗
Research note: effect of duodenal--infused unsaturated fatty acids on dairy milk composition.
Investigations were conducted to assess the effect of duodenal infusions of oleic and linoleic acids both on milk composition and fatty acid patterns of milk fat. The investigations were carried out in two trials, I and II, each with 3 cows, fitted with a ruminal and a duodenal cannula. 56 g of oleic acid (Trial I) and 46 g linoleic acid (Trial II) were infused daily into the duodenum over a period of 7 days, while no application of fatty acids acted as a control in each case. An increase in milk fat content was observed in both trials whereas the content of protein and lactose in milk remained unaffected. In both trials the milk oleic acid and linoleic acid concentrations increased. In trial I C18:1 increased from 14.8 to 18.7%. In trial II C18:2 increased from 2.5 to 5.8% of total lipids. It was estimated that 84.8% of the infused C18:1 and 86.5% of C18:2 were transferred into milk fat.
Animals↗
Sequential changes of biochemical bone parameters after kidney transplantation.
BACKGROUND: Persistent hyperparathyroidism after renal transplantation (Rtx) has been reported in several studies. However these studies evaluated biochemical bone parameters either only during a short time period (up to 6 months) or for a longer time period, but with long intervals in between. Therefore, we prospectively evaluated biochemical bone parameters of kidney-transplant recipients at short intervals for 2 years after surgery. METHODS: Biochemical bone parameters were prospectively investigated in 129 patients 2, 3, 5, 8, 12, 18 and 24 months after Rtx. All patients received prednisone and cyclosporin A as immunosuppressive therapy, and 75 patients also received azathioprine. None of the patients was treated with calcium, phosphorus, or vitamin D preparations. RESULTS: Serum creatinine levels decreased from 166.8 +/- 5.4 mumol/l to 140.0 +/- 4.9 two years after Rtx; (data are expressed as mean +/- s.e.m.). Serum phosphorus levels increased slightly from 0.9 +/- 0.022 mmol/l to 0.98 +/- 0.025 (12 m), but remained within the lower normal range. We observed a rise in total and albumin adjusted calcium concentrations 3 months after Rtx. 52% of all patients had serum calcium levels above 2.62 mmol/l (upper normal limit in our laboratory) 3 months after renal transplantation with a gradual decrease thereafter. There was no correlation of calcium and PTH levels. We observed a significant rise in biochemical bone parameters from 2 to 5 months after renal transplantation (P < 0.001): alkaline phosphatase (AP) increased from 164.3 +/- 9.4 to 236 +/- 12.7 U/l (normal 50-180), bone specific alkaline phosphatase (BAP) rose from 17.7 +/- 1.36 to 23.2 +/- 1.7 ng/ml (normal:4-20) and osteocalcin (OC) increased from 20.2 +/- 1.5 to 26.7 +/- 1.9 ng/ml (normal 4-12). AP and BAP levels values normalized 12 months after renal transplantation, whereas OC was still above normal throughout the study period. Patients were subdivided into two groups: those with good and those with impaired graft functions. Patients with good graft function had stable serum creatinine levels (< or = 132 mumol/l or < or = 1.5 mg/dl) well below the mean serum creatinine concentration during the study period. The significant changes in AP, BAP, and OC occurred irrespective of renal function. However, patients with impaired graft function (n = 65) had significantly higher PTH-levels (70 pg/ml higher) than patients with good graft function (n = 64), P < 0.01. PTH was positively correlated with serum creatinine (r = 0.81, P < 0.001). Moreover, patients with low 25 (OH) vitamin D levels (n = 63) had significantly higher PTH concentrations (between 40 and 80 pg/ml, P < 0.01) throughout the study period compared to patients (n = 66) with a sufficient 25(OH)D supply irrespective of graft function. There was a negative correlation of 25 (OH)D levels and PTH; (r = -0.49, P < 0.001). 1,25(OH)2D3 (evaluated in 24 patients) levels increased from 46.5 +/- 6.6 to 76.9 +/- 7.6 pg/ml (normal:35-90) at 12 months. CONCLUSION: Hypercalcaemia is a common phenomenon in the early period after kidney transplantation and occurs in the presence of low normal phosphorus levels. It is most probably related to improved PTH action and 1-hydroxylation of vitamin D. The rise in biochemical bone parameters between 3 and 5 months occurs irrespective of graft function and normalization is only achieved 1 year after transplantation. PTH is constantly elevated for up to 2 years after kidney transplantation and is most probably related (a) to impaired graft function and (b) to suboptimal 25 OH vitamin D supply.
Adult↗