How to recognise inborn errors of metabolism.
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Family Practice↗
Biomedical subjects
J Walter
Publications and source records attributed to J Walter.
M.phi 3TII: a new monospecific DNA (cytosine-C5) methyltransferase with pronounced amino acid sequence similarity to a family of adenine-N6-DNA-methyltransferases.
The temperate B.subtilis phages phi 3T and rho 11s code, in addition to the multispecific DNA (cytosine-C5) methyltransferases (C5-MTases) M. phi 3TI and M. rho 11sI, which were previously characterized, for the identical monospecific C5-MTases M. phi 3TII and M. rho 11sII. These enzymes modify the C of TCGA sites, a novel target specificity among C5-MTases. The primary sequence of M. phi 3TII (326 amino acids) shows all conserved motifs typical of the building plan of C5-MTases. The degree of relatedness between M. phi 3TII and all other mono- or multispecific C5-MTases ranges from 30-40% amino acid identity. Particularly M. phi 3TII does not show pronounced similarity to M. phi 3TI indicating that both MTase genes were not generated from one another but were acquired independently by the phage. The amino terminal part of the M. phi 3TII (preceding the variable region 'V'), which predominantly constitutes the catalytic domain of the enzyme, exhibits pronounced sequence similarity to the amino termini of a family of A-N6-MTases, which--like M.TaqI--recognize the general sequence TNNA. This suggests that recently described similarities in the general three dimensional organization of C5- and A-N6-MTases imply divergent evolution of these enzymes originating from a common molecular ancestor.
Amino Acid Sequence↗
M.phi 3TII: a new monospecific DNA (cytosine-C5) methyltransferase with pronounced amino acid sequence similarity to a family of adenine-N6-DNA-methyltransferases.
The temperate B.subtilis phages phi 3T and rho 11s code, in addition to the multispecific DNA (cytosine-C5) methyltransferases (C5-MTases) M.phi 3TI and M.rho 11sI, which were previously characterized, for the identical monospecific C5-MTases M.phi 3TII and M.rho 11sII. These enzymes modify the C to TCGA sites, a novel target specificity among C5-MTases. The primary sequence of M.phi 3TII (326 amino acids) shows all conserved motifs typical of the building plan of C5-MTases. The degree of relatedness between M.phi 3TII and all other mono- or multispecific C5-MTases ranges from 30-40% amino acid identity. Particularly M.phi 3TII does not show pronounced similarity to M.phi 3TI indicating that both MTase genes were not generated from one another but were acquired independently by the phage. The amino terminal part of the M.phi 3TII (preceding the variable region 'V'), which predominantly constitutes the catalytic domain of the enzyme, exhibits pronounced sequence similarity to the amino termini of a family of A-N6-MTases, which--like M.Taql--recognize the general sequence TNNA. This suggests that recently described similarities in the general three dimensional organization of C5- and A-N6-MTases imply divergent evolution of these enzymes originating from a common molecular ancestor.
Amino Acid Sequence↗
Two homeo domain proteins bind with similar specificity to a wide range of DNA sites in Drosophila embryos.
We have used in vivo UV cross-linking to directly measure DNA binding by the homeo domain proteins even-skipped (eve) and fushi tarazu (ftz) in Drosophila embryos. Strikingly, these two proteins bind at uniformly high levels throughout the length of their genetically identified target genes and at lower, but significant, levels to genes that they are not expected to regulate. The data also suggest that these two proteins have very similar DNA-binding specificities in vivo. In contrast, a non-homeo domain transcription factor, zeste, is only detected on short DNA elements within a target promoter and not on other genes. These results are consistent with the in vitro properties of these various proteins, their respective concentrations in the nucleus, and with earlier predictions of how transcription factors bind DNA in vivo. We propose that these data favor the model that eve, ftz, and closely related homeo domain proteins act by directly regulating mostly the same target genes.
Animals↗
Methylation analysis on individual chromosomes: improved protocol for bisulphite genomic sequencing.
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5-Methylcytosine↗
Anatomical urinary stress incontinence in women with rheumatoid arthritis: its frequency and coping strategies.
OBJECTIVE: The objectives of this study were to develop a valid screening tool for anatomical urinary stress incontinence (AUSI), to use it to determine the frequency of AUSI in women with RA, and to identify the cognitive, behavioral, and psychological coping strategies used by these women to deal with incontinence. METHODS: A screening tool concerning AUSI was validated by comparing self-reported incidences of AUSI with clinical evaluation. Questionnaires regarding presence of AUSI and cognitive, behavioral, and psychological coping strategies were sent to 750 women clinically diagnosed with RA. RESULTS: Of the 262 respondents (35% response rate), 21% had characteristics of AUSI, a rate similar to general population studies. Coping strategies varied depending on whether the women were at home or away from home and whether the women were alone or with others. CONCLUSIONS: There is a need for women with incontinence and the health care workers who serve them to be educated about AUSI and its treatments.
Adaptation, Psychological↗
Protein purification: aspects of processes for pharmaceutical products.
The concept of purity is addressed for proteins as drugs. In addition to a short description of general techniques, emphasis is given to a discussion of special protein impurities and contaminants from the host cell of process additives. This includes the host homologue to the product and biologically active molecules and the quantitation of host cell proteins. Of special concern are closely related non functional proteins from the gene for the protein product itself. They pose the question of how a 'pure' protein can be defined, and to what extent potential heterogeneities caused by the purification method represent a risk. Most relevant are 'invisible' contaminants such as potential virus including the BSE agent. Critical aspects will be discussed of the current rationale for a strategy to arrive at safe biological products. The limitations of current virus enumeration methods are discussed in relation to technical limitations to prove the efficiency of virus removal steps. Another limitation is the nature of the protein product and its individual resistance to denaturing conditions in virus inactivation steps. This is amply demonstrated by the lack of methods for inactivation of the BSE agent maintaining the native state of proteins. As a general recommendation it is proposed to base risk assessment on scientific data and come to scientifically defined criteria of acceptance in both the global and national communities. This will facilitate bringing new biopharmaceuticals to the benefit of patients in the shortest period of time.
Animals↗
Symptomatic hereditary type-II protein C deficiency caused by a missense mutation in exon IX of the protein C gene (Gly381 to Ser).
We report the characterization of the genetic defect in a family with hereditary type-II protein C (PC) deficiency. The propositus is a 28-year-old woman with a history of thrombosis. Her PC activity level (58%) and PC antigen level (115%) are compatible with the diagnosis of type-II PC deficiency. Her asymptomatic sister is also PC deficient. Analysis of the PC gene of the propositus revealed a point mutation (G to A) at nucleotide 8856, which results in the replacement of Gly381 by Ser in the heavy chain of PC. The amino acid change occurs close to the active-site serine at a residue which is highly conserved among the serine proteases. The mutation is also present in the PC gene of the propositus' sister. Her brother, who is asymptomatic, has a normal genotype with respect to the mutation at nucleotide 8856.
Adult↗
Estimation of cerebral blood flow through color duplex sonography of the carotid and vertebral arteries in healthy adults.
BACKGROUND AND PURPOSE: To noninvasively estimate cerebral blood flow volume, a prospective study of color duplex sonography of the common, external, and internal carotid arteries and vertebral arteries of healthy adults was done. Cerebral blood flow was calculated with the sum of flow volumes in the internal carotid and vertebral arteries of both sides. METHODS: Using a 7.0-MHz linear transducer of a computed sonography system, cervical arteries of 48 volunteers (23 women, 25 men; mean age, 35 +/- 12 years) were examined. We measured angle-corrected time-averaged velocities and the diameter of the vessels and calculated the flow volumes of all arteries. In addition, peak systolic, maximum end-diastolic, and time-averaged maximum velocities and the resistance, pulsatility, and spectral broadening indexes were determined. Furthermore, we analyzed the side-to-side difference, age dependence, and long-term reproducibility of these parameters. RESULTS: The mean +/- SD values of flow volumes in the common, internal, and external carotid and vertebral arteries were 470 +/- 120, 265 +/- 62, 160 +/- 66, and 85 +/- 33 mL/min on either side, respectively. Total cerebral blood flow was 701 +/- 104 mL/min (corresponding to 54 +/- 8 mL/100 g per minute), with no variation in age or sex. Long-term reproducibility of cerebral blood flow and flow volumes in all vessels was significant (P < .01). CONCLUSIONS: We conclude that color duplex sonography of cervical arteries is potentially a practical method for estimating total cerebral blood flow. This noninvasive technique may be ideally suited for bedside and follow-up examinations of the critically ill patient. In future studies it should be compared with established radionuclide techniques.
Adult↗
Developmental control of allelic methylation in the imprinted mouse Igf2 and H19 genes.
The Insulin-like growth factor 2 (Igf2) and H19 genes are reciprocally imprinted and closely linked. Igf2 encodes a fetal growth-factor and is predominantly expressed from the paternal allele, while H19 is expressed from the maternal allele and encodes a transcript which may downregulate cellular proliferation. One of the epigenetic modifications thought to be involved in parental imprinting is DNA methylation. Here we analyse methylation in two regions of the Igf2 gene, one approx. 3 kb upstream of the gene and one in the 3' part of the gene. Both regions are more methylated on the expressed paternal chromosome. Genomic sequencing of individual chromosomes in the first region shows this parent-specific methylation to be highly mosaic; interestingly, individual sperm chromosomes carry different methylation patterns into the egg. In the more 3' region, which is fully methylated in sperm, the level of methylation on the paternal allele is highly tissue-specific and is correlated with expression of the gene in fetal tissues. Hence, the paternal allele is highly methylated in fetal liver (high expression) but is undermethylated in fetal brain (virtually no expression). Adult choroid plexus, a brain tissue in which Igf2 is expressed from both alleles and H19 is not expressed, represents an apparent loss of imprinting. Here, both Igf2 and H19 adopt a paternal type methylation pattern on both parental chromosomes. Analysis of early-passage androgenetic and parthenogenetic embryonic stem (ES) cells shows that the methylation patterns of Igf2 and H19 on maternal and paternal chromosomes are very similar. Androgenetic and parthenogenetic teratomas derived from these ES cells show the appropriate paternal and maternal patterns, respectively, of allelic methylation in both genes. Our results suggest that allelic methylation patterns in Igf2 and H19 arise early in embryogenesis and change progressively during development. Some of these developmental changes are apparently under tissue-specific control.
Alleles↗
Evidence for CKI and CKII at the cell surface.
Ser/Thr-protein kinases at the cell surface (ecto-PK) use physiological concentrations of extracellular ATP for phosphorylation of endogenous cell surface proteins, as well as of soluble protein substrates in the extracellular environment (Kübler et al., 1982, 1989). One abundant ecto-PK component is believed to be a protein kinase CKII since it phosphorylates phosvitin and casein, is sensitive to heparin at low concentrations, and can use both ATP and GTP as cosubstrate. This ecto-PK activity can be detached from the surface of intact cells through interaction with exogenous substrates, a process termed "shedding" (Kübler et al., 1983). This study reports a method for the purification and identification of shedded ecto-PK. Affinity chromatography of the concentrated ecto-PK through a heparin-matrix resolved two phosvitin/casein kinase activities upon elution with a NaCl gradient, termed as peak I and peak II. Relative to the total protein load of the cells employed for ecto-PK shedding, the specific activities increased by a factor of about 10(4) times. The use of peptide substrates specific for CKI and CKII, of ATP and GTP, as well as of antibodies specific for CKII subunit, clearly identified one of the enzymes as a CKI-like entity and the other one as CKII-like. Although the spatial arrangement on the cell surface of the two related ecto-PKs is unknown, their tandem appearance together in the cell supernatant might suggest the possibility of a functional unit.
Adenosine Triphosphate↗
Six novel and three recurrent mutations in nine Austrian patients with hemophilia B.
In this report we describe the molecular basis of the factor IX (FIX) deficiency in nine patients with severe (n = 6), moderate (n = 1) or mild (n = 2) hemophilia B. The following genetic defects were identified by enzymatic amplification with the polymerase chain reaction (PCR) and subsequent direct sequencing of all exons and exon-intron-junctions: patient B.B. (FIX "Vienna I"): deletion of nucleotides 6343 to 6362; patient M.H. and W.J. (FIX "Vienna II"): nucleotide 17704 (C to G), Gln 97 to Glu; patient L.K. (FIX "Vienna III"): nucleotide 17761 (C to T), Arg 116 to stop; patient U.A. (FIX "Vienna IV"): nucleotide 10415 (C to G), Pro 55 to Ala; patient H.G. (FIX "Vienna V"): nucleotide 6488 (C to T), Thr 38 to Ile; patient H.M. (FIX "Vienna VI"): nucleotide 31276 (G to C), Trp 385 to Cys; patient L.C. (FIX "Vienna VII"): deletion of nucleotide 6700; patient S.F. (FIX "Vienna VIII"): nucleotide 10392 (A to T), Asp 47 to Val. The causative mutation was detected in the FIX gene in each of the nine patients with hemophilia B. There was one small deletion, one point deletion and seven point mutations. The latter include six missense mutations and one nonsense mutation. The mutations in Vienna III, IV and V have already been described in previous studies. The two deletions, Vienna I and Vienna VII have not been reported previously. The genetic defects observed in Vienna II, VI and VIII are novel missense mutations which result in amino acid changes at residues 97, 47 and 385, respectively.
Austria↗
Molecular and functional basis of hereditary factor IX defects.
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DNA Mutational Analysis↗
Transcranial color duplex sonography in childhood and adolescence. Age dependence of flow velocities and waveform parameters.
BACKGROUND AND PURPOSE: Transcranial color duplex sonographic examinations in children and adolescents without cerebrovascular disease were evaluated retrospectively. Flow velocities and waveform parameters were determined and their side-to-side differences and age dependence analyzed and, finally, compared with analogous data of a previously described group of healthy adults. METHODS: With a 2.0-MHz sector transducer of a computed sonographic system, the anterior, middle, and posterior cerebral arteries were examined in 64 children and adolescents between 1.5 and 17.5 years of age. Angle-corrected systolic peak, end-diastolic maximum, time-averaged, and time-averaged maximum velocities, and the resistance, pulsatility, and spectral broadening indexes were determined in all vessels. RESULTS: Mean +/- SD values for time-averaged maximum velocity (and time-averaged velocity) were 92.2 +/- 13.0, 79.9 +/- 17.7, and 63.9 +/- 13.6 (55.3 +/- 11.7, 40.4 +/- 10.4, and 34.2 +/- 9.2) cm/s, respectively, in the middle, anterior, and posterior cerebral arteries in children under 10 years of age; values were 83.2 +/- 11.9, 69.4 +/- 13.8, and 55.6 +/- 10.1 (50.8 +/- 9.0, 39.9 +/- 10.5, and 33.1 +/- 6.3) cm/s, respectively, in children 10 years of age and older. Time-averaged maximum velocity decreased significantly with age in all vessels (P < .001). Although time-averaged velocity did not change significantly during childhood and adolescence, a clear decline occurred from adolescence to adulthood (P < .0001 in the middle and posterior cerebral arteries; P < .01 in the anterior cerebral artery). The spectral broadening index decreased significantly from childhood to adolescence in the anterior and posterior cerebral arteries (P < .0001). The resistance and pulsatility indexes remained stable throughout childhood. CONCLUSIONS: Transcranial color duplex sonography allows angle-corrected measurements of "true" flow velocities in basal cerebral arteries. Additional determination of time-averaged velocity permits more detailed evaluation of flow characteristics for all age groups. The transcranial color duplex technique may provide deeper insights on normal cerebral perfusion and its disorders.
Adolescent↗
Comparative study of transcranial color duplex sonography and transcranial Doppler sonography in adults.
To determine whether the frequency shift recorded in basal cerebral arteries corresponds to "true" flow velocities, a prospective comparative study of transcranial color duplex sonography (TCCD) and transcranial Doppler sonography (TCD) was performed. A 2.0-MHz transducer of a computerized TCCD system and a TCD device were used. The middle cerebral artery (MCA) and anterior cerebral artery (ACA) were examined by TCCD in 49 healthy volunteers (mean age 35 +/- 12 years). In 45 of the same volunteers a comparative TCD examination was possible. The studies were carried out blindly by different examiners at separate appointments. Peak systolic flow velocity, end-diastolic maximum flow velocity, time-averaged maximum flow velocity, and the pulsatility index were measured by both techniques. Additionally, for TCCD, time-averaged flow velocity was assessed, the resistance index and a spectral broadening index were calculated, and the energy output required for reliable measurement was analyzed. The TCCD signals were recorded in 98% of both MCA's and ACA's; with TCD, signals were recorded in 98% of MCA's and 87% of ACA's. Although in both vessels the angle-corrected peak systolic and time-averaged maximum velocities were approximately 10% to 15% higher in TCCD than in TCD measurements, correlation of flow velocities between both techniques was significant (p < 0.0001); differences between sides and age-dependency of flow velocities corresponded as well. In a reproducibility study, TCCD was repeated in 27 subjects by a third examiner with significant correlation (p < 0.0001) of both TCCD examinations. It is concluded that the advantage of TCCD is associated more with a qualitative aspect than a quantitative one. The additional visual dimension of TCCD can open new diagnostic possibilities in cerebrovascular disorders.
Adult↗
Does captopril decrease the effect of human recombinant erythropoietin in haemodialysis patients?
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Captopril↗
BsuBI--an isospecific restriction and modification system of PstI: characterization of the BsuBI genes and enzymes.
The enzymes of the Bacillus subtilis BsuBI restriction/modification (R/M) system recognize the target sequence 5'CTGCAG. The genes of the BsuBI R/M system have been cloned and sequenced and their products have been characterized following overexpression and purification. The gene of the BsuBI DNA methyltransferase (M.BsuBI) consists of 1503 bp, encoding a protein of 501 amino acids with a calculated M(r) of 57.2 kD. The gene of the restriction endonuclease (R.BsuBI), comprising 948 bp, codes for a protein of 316 amino acids with a predicted M(r) of 36.2 kD. M.BsuBI modifies the adenine (A) residue of the BsuBI target site, thus representing the first A-N6-DNA methyltransferase identified in B. subtilis. Like R.PstI, R.BsuBI cleaves between the A residue and the 3' terminal G of the target site. Both enzymes of the BsuBI R/M system are, therefore, functionally identical with those of the PstI R/M system, encoded by the Gram negative species Providencia stuartii. This functional equivalence coincides with a pronounced similarity of the BsuBI/PstI DNA methyltransferases (41% amino acid identity) and restriction endonucleases (46% amino acid identity). Since the genes are also very similar (58% nucleotide identity), the BsuBI and PstI R/M systems apparently have a common evolutionary origin. In spite of the sequence conservation the gene organization is strikingly different in the two R/M systems. While the genes of the PstI R/M system are separated and transcribed divergently, the genes of the BsuBI R/M system are transcribed in the same direction, with the 3' end of the M gene overlapping the 5' end of the R gene by 17 bp.
Amino Acid Sequence↗
Local growth of a Burkitt's lymphoma versus disseminated invasive growth of the autologous EBV-immortalized lymphoblastoid cells and their somatic cell hybrids in SCID mice.
Specific host-graft interactions, as well as intrinsic properties of transferred cell, determine tumorigenicity in xenogeneic systems. We compared the growth characteristics of human B-lymphoid cell lines in SCID mice with the well characterized growth pattern in nude mice and observed striking differences in malignancy in the respective hosts. Two cell lines derived from the same individual, the Epstein-Barr-virus(EBV)-positive Burkitt's lymphoma BL 60 (BL) and the autologous EBV-immortalized lymphoblastoid cell line IARC 277 (LCL) were used. In addition, we tested somatic cell hybrids (HYB) of both cell lines, which despite the LCL-like differentiation phenotype show the de-regulated c-myc expression pattern of the parental BL line, assumed to be a critical factor in BL pathogenesis. Subcutaneously (s.c.) injected BL cells produced local progressively growing tumor masses at the injection site without distant metastases in both nude and SCID mice. Although both mouse strains possess the same genetic background (BALB/c) and differ only in the B-cell sub-set, the growth patterns of the LCL and hybrids were completely different. In contrast to the regressive behaviour of LCL and hybrids in nude mice, these lines show invasive and disseminated progressive growth in SCID mice. Peripheral lymph nodes an thymic tissue were preferentially colonized, whereas mucosal-associated lymphoid tissue (Peyer's patches and appendix) and spleen were not infiltrated. The preferential migration of lymphocytes to certain tissues is termed homing in a syngeneic system and mediated by homing receptors and vascular addressins. The "homing" of LCL and hybrids into lymphoid SCID mouse tissue suggests a strong interaction with the endothelial cells of the host. Detailed phenotypic analysis of BL, LCL and 3 different hybrids was performed using an antibody panel against differentiation and adhesion markers. Overall dominance of the LCL phenotype was observed in the hybrids, as indicated by cytology, tumor growth, dissemination and the pattern of surface-marker expression. The c-myc activation in hybrids does not appear to influence growth behavior.
Animals↗