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PubMed1992

Transforming properties and substrate specificities of the protein tyrosine kinase oncogenes ros and src and their recombinants.

To determine the sequences of the oncogenes src (encoded by Rous sarcoma virus [RSV]) and ros (encoded by UR2) that are responsible for causing different transformation phenotypes and to correlate those sequences with differences in substrate recognition, we constructed recombinants of the two transforming protein tyrosine kinases (PTKs) and studied their biological and biochemical properties. A recombinant with a 5' end from src and a 3' end from ros, called SRC x ROS, transformed chicken embryo fibroblasts (CEF) to a spindle shape morphology, mimicking that of UR2. Neither of the two reverse constructs, ROS x SRC I and ROS x SRC II, could transform CEF. However, a transforming variant of ROS x SRC II appeared during passages of the transfected cells and was called ROS x SRC (R). ROS x SRC (R) contains a 16-amino-acid deletion that includes the 3' half of the transmembrane domain of ros. Unlike RSV, ROS x SRC (R) also transformed CEF to an elongated shape similar to that of UR2. We conclude that distinct phenotypic changes of RSV- and UR2-infected cells do not depend solely on the kinase domains of their oncogenes. We next examined cellular proteins phosphorylated by the tyrosine kinases of UR2, RSV, and their recombinants as well as a number of other avian sarcoma viruses including Fujinami sarcoma virus Y73, and some ros-derived variants. Our results indicate that the UR2-encoded receptorlike PTK P68gag-ros and its derivatives have a very restricted substrate specificity in comparison with the nonreceptor PTKs encoded by the rest of the avian sarcoma viruses. Data from ros and src recombinants indicate that sequences both inside and outside the catalytic domains of ros and src exert a significant effect on the substrate specificity of the two recombinant proteins. Phosphorylation of most of the proteins in the 100- to 200-kDa range correlated with the presence of the 5' src domain, including the SH2 region, but not with the kinase domain in the recombinants. This corroborates the conclusion given above that the kinase domain of src or ros per se is not sufficient to dictate the transforming morphology of these two oncogenes. High-level tyrosyl phosphorylation of most of the prominent substrates of src is not sufficient to cause a round-shape transformation morphology.

Amino Acid Sequence↗

PubMed2006

Intrahepatic cholestasis of pregnancy: the severe form is associated with common variants of the hepatobiliary phospholipid transporter ABCB4 gene.

BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) is characterised by troublesome maternal pruritus, raised serum bile acid levels and increased fetal risk. Mutations of the ABCB4 gene encoding the hepatobiliary phospholipid transporter have been identified in a small proportion of patients with cholestasis of pregnancy. In a recent prospective study on 693 patients with cholestasis of pregnancy, a cut-off level for serum bile acid (> or =40 micromol/l) was determined for increased risk of fetal complications. OBJECTIVES: To investigate whether common combinations of polymorphic alleles (haplotypes) of the genes encoding the hepatobiliary ATP-binding cassette (ABC) transporters for phospholipids (ABCB4) and bile acids (ABCB11) were associated with this severe form of cholestasis of pregnancy. METHODS: For genetic analysis, 52 women with bile acid levels > or =40 micromol/l (called cases) and 52 unaffected women (called controls) matched for age, parity and geographical residence were studied. Gene variants tagging common ABCB4 and ABCB11 haplotypes were genotyped and haplotype distributions were compared between cases and controls by permutation testing. RESULTS: In contrast with ABCB11 haplotypes, ABCB4 haplotypes differed between the two groups (p = 0.019), showing that the severe form of cholestasis of pregnancy is associated with the ABCB4 gene variants. Specifically, haplotype ABCB4_5 occurred more often in cases, whereas haplotypes ABCB4_3 and ABCB4_7 were more common in controls. These associations were reflected by different frequencies of at-risk alleles of the two tagging polymorphisms (c.711A: odds ratio (OR) 2.27, p = 0.04; deletion intron 5: OR 14.68, p = 0.012). CONCLUSION: Variants of ABCB4 represent genetic risk factors for the severe form of ICP in Sweden.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2005

Biochemical characteristics of variant transthyretins causing hereditary leptomeningeal amyloidosis.

Transthyretin (TTR) is a tetrameric protein that can dissociate into amyloidogenic monomers and cause TTR-related amyloidosis. A rare phenotype, called hereditary leptomeningeal TTR amyloidosis, in which TTR amyloid deposition occurs mainly in leptomeninges and subarachnoid vessels, has been reported in patients with several different TTR variants. In the present study, we examined TTR variants immunoprecipitated from the serum and cerebrospinal fluid (CSF) of patients with hereditary leptomeningeal TTR amyloidosis using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (IP-Mass method). The leptomeningeal-type TTR variants were not detected in the serum but were found at low levels in the CSF. The undetectable levels of the leptomeningeal-type TTR variants in serum could explain the minute amounts of systemic deposition of these variants. The relatively high level of unstable TTR variants in CSF, probably due to increased secretion from the choroid plexus, is considered to be the pathogenesis of the leptomeningeal-type of TTR amyloidosis.

Adult↗

PubMed2006

Single potential electrophoresis microchip with reduced bias using pressure pulse injection.

We propose two variants of a new injection technique for use in electrophoresis microchips, called "front gate pressure injection" and "back gate pressure injection", that both enable a controlled and reproducible sample introduction with reduced bias compared to electrokinetic gated injection. A continuous flow of a test solution of fluorescein/rhodamine B in 20 mM Tris/boric acid buffer (pH 8.6) sample test solution is electrokinetically driven near to the entrance of the separation channel, using a single voltage (3 kV) that is constant in time. A sample plug is injected in the separation channel by a pressure pulse of the order of 0.1 s. The latter is generated using the mechanical deflection of a PDMS membrane that is loosely placed on a dedicated chip reservoir. The analysis of the peak area ratio of the separated compounds demonstrates a nearly constant sample composition when using pressure-based injection. A small remaining injection bias for the shortest membrane deflection times can be attributed to a dilution effect of the charged compound due to the presence of an electrical field transverse to the sample flow boundary in the channel junction.

Dimethylpolysiloxanes↗

PubMed2001

The molecular physiology of the cardiac transient outward potassium current (I(to)) in normal and diseased myocardium.

G. Y. Oudit, Z. Kassiri, R. Sah, R. J. Ramirez, C. Zobel and P. H. Backx. The Molecular Physiology of the Cardiac Transient Outward Potassium Current (I(to)) in Normal and Diseased Myocardium. Journal of Molecular and Cellular Cardiology (2001) 33, 851-872. The Ca(2+)-independent transient outward potassium current (I(to)) plays an important role in early repolarization of the cardiac action potential. I(to)has been clearly demonstrated in myocytes from different cardiac regions and species. Two kinetic variants of cardiac I(to)have been identified: fast I(to), called I(to,f), and slow I(to), called I(to,s). Recent findings suggest that I(to,f)is formed by assembly of K(v4.2)and/or K(v4.3)alpha pore-forming voltage-gated subunits while I(to,s)is comprised of K(v1.4)and possibly K(v1.7)subunits. In addition, several regulatory subunits and pathways modulating the level and biophysical properties of cardiac I(to)have been identified. Experimental findings and data from computer modeling of cardiac action potentials have conclusively established an important physiological role of I(to)in rodents, with its role in large mammals being less well defined due to complex interplay between a multitude of cardiac ionic currents. A central and consistent electrophysiological change in cardiac disease is the reduction in I(to)density with a loss of heterogeneity of I(to)expression and associated action potential prolongation. Alterations of I(to)in rodent cardiac disease have been linked to repolarization abnormalities and alterations in intracellular Ca(2+)homeostasis, while in larger mammals the link with functional changes is far less certain. We review the current literature on the molecular basis for cardiac I(to)and the functional consequences of changes in I(to)that occur in cardiovascular disease.

Action Potentials↗

PubMed1979

Clinical, morphological, and biochemical investigations on a patient with an unusual form of neuronal ceroid-lipofuscinosis.

A patient with a progressive neurological disorder beginning at the age of three years is described. Mental and visual disturbances were the first signs, soon followed by ataxia and myoclonic jerks. Fundoscopy revealed a decreased pigmentation of the retina. Ultramicroscopic investigations of muscle and skin disclosed the typical changes seen in the late infantile and juvenile forms of neuronal ceroid-lipofuscinosis. In contrast to the clinical and ultrastructural findings, the fatty acid pattern of the serum lecithin showed a significant increase of arachidonic acid and a corresponding decrease of linoleic acid which is characteristic of the so-called infantile form of neuronal ceroid-lipofuscinosis (Hagberg-Santavuori variant; polyunsaturated fatty acid lipidosis). The obvious heterogeneity of the clinical, histological and laboratory findings within the subgroups of neuronal ceroid-lipofuscinosis is briefly discussed.

Ataxia↗

PubMed1987

Abdominal tuberculosis.

The recent literature on abdominal tuberculosis is comprehensively reviewed, and seven cases of abdominal tuberculosis are reported, including four belonging to three generations of the same family. Possible explanations for this familial incidence are discussed. Abdominal tuberculosis is not so rare; 135 cases have recently been reported from the United States and Canada. This review dissipates four common misconceptions: abdominal tuberculosis is rare, tuberculosis is a stigmata of the poor, abdominal tuberculosis is always associated with active pulmonary tuberculosis, and chronic abdominal pathology is synonymous with regional enteritis. Since the description of regional enteritis, more and more cases of chronic intestinal pathology have been labeled "regional enteritis." The fact that intestinal tuberculosis is rather uncommon should not automatically lead to the diagnosis of regional enteritis. The possibility that many cases of so-called regional enteritis may, in fact, be a stage or a variant of abdominal tuberculosis, is worth considering. Abdominal tuberculosis is not a relic of the past. It remains a real challenge to the diagnostic acumen and therapeutic skills of both the internist and the surgeon.

Abdomen↗

PubMed2005

A founder mutation in the CLCNKB gene causes Bartter syndrome type III in Spain.

The term "Bartter syndrome" encompasses a group of closely related inherited tubulopathies characterized by markedly reduced NaCl transport by the distal nephron. At present, five different genetic variants have been demonstrated. The majority of patients with so-called classic Bartter syndrome carry inactivating mutations of the CLCNKB gene encoding the basolateral ClC-Kb chloride channel (Bartter syndrome type III). The purpose of this study was to investigate the underlying mutation in cases of classic Bartter syndrome followed at our center. Ten patients, including two sisters, with clinical and biochemical features of classic Bartter syndrome were included in the mutational analysis. They originated from different regions of Spain with either Basque or Spanish ancestry. There was no history of consanguineous marriage in any of the kindreds. The parents and siblings of each patient, as well as a population of 300 healthy control adult subjects, were also analyzed. All ten patients were found to be homozygous for an identical missense mutation in the CLCNKB gene, substituting a threonine for an alanine at codon 204 (A204T) in the putative fifth transmembrane domain of the protein. None of the 300 control subjects were homozygous for the A204T allele. Overall, the A204T mutation was detected on 2/600 control chromosomes. Despite sharing a common mutation, the clinical manifestations of the syndrome in the patients varied from lack of symptoms to severe growth retardation. Demonstration of a point mutation within the CLCNKB gene as the apparently unique cause of Bartter syndrome type III in Spain is highly suggestive of a founder effect. Our results also support the lack of correlation between genotype and phenotype in this disease.

Adolescent↗

PubMed1991

DNA typing for class II HLA antigens with allele-specific or group-specific amplification. IV. Typing for alleles of the HLA-DR2 group.

In this study we present an approach for the definition of the alleles belonging to the HLA-DR2 group by DNA typing with oligonucleotide probes. Following methodology similar to that we used previously for the definition of other HLA-DR subsets, we have now developed primers for DR2-DRB1 and DR2-DRB5 amplification, and probes for the identification of sequences that distinguish the subtypes of this group of genes. The method used defines all the previously described alleles at both DR2-associated DRB loci. In addition, we have identified a variant of DRB1-DR2-Dw2. This new allele has been called DRB1*15.3. It is different from DRB1*1501 in codon 30, where it carries histidine instead of tyrosine. Eight different haplotype combinations of DRB5, DRB1, and DQB1 were identified within the DR2 group and their occurrence in four normal panels of different ethnic origin has been described. Haplotypes containing DRB1*15.3 occurred most frequently in black panel members in whom it was associated with either DQB1*0602 or DQB1*0501. Two unusual haplotypes were observed: one containing elements of DR2-Dw21 (DQB1*0502) and of DR2-Dw22 (DRB1*1602) and one containing elements of Dw21 (DRB1*1601, DQB1*0502) and Dw2 (DRB5*0101). The methods described permit simple and rapid determination of the alleles of the HLA-DR2 group and should be useful for population studies and for investigation of DR2-associated diseases.

Alleles↗

PubMed1992

HLA polymorphism in a Mataco South American Indian tribe: serology of class I and II antigens. Molecular analysis of class II polymorphic variants.

In the present study, HLA-A, B, C, DR, DQ, and DP loci were analyzed in a group of Mataco Amerindians of Argentina. Using reagents from the 11th International Histocompatibility Workshop (11th IHW), class I specifities such as Bw70, Bw75, and Bw48 were found in this population, other than the HLA determinants commonly described in South American Indians. The class II antigens found were DR4, DRw14, and DRw8 at the DR locus, and DQw4 and DQw7 at the DQ locus. The analysis of DRB1-DR4 related alleles, performed by PCR amplification and oligonucleotide probe hybridization, showed the presence of DRB1*0403, *0404, *0405, and *0411 in individuals from this ethnic group. By the analysis of DRB1-DRw14 related alleles, two variants were found: DRB1*1402 and DRB1*1406, the latter provisionally called DRB1 14.6 in 11th IHW. The DRw8-related allele present was DRB1*0802. The analysis of DRB3 gene revealed only the presence of DRB3*0101 allele in DRw14 individuals. DPB1 locus was also analyzed in unrelated individuals of the same population. Only five DPB1 alleles were found: DPB1*0201, *0301, *0402, *0501, and *1301 over the 19 previously described in the literature. These findings emphasize the restricted HLA class I and II variation observed in this ethnic group as it has been previously shown in other American groups. Some particular haplotypes in this Mataco tribe are described in this work.

Alleles↗

PubMed2005

Purine activity of RNase T1RV is further improved by substitution of Trp59 by tyrosine.

Ribonuclease T1 is an enzyme that cleaves single-stranded RNA with high specificity after guanylyl residues. Although this enzyme is a very good characterized protein with respect to structure and enzymatic function, we were only recently successful in generating RNase T1-RV, a variant where the specificity was changed from guanine to purine. As this change of substrate specificity was made at the cost of activity, the aim was now to further improve the overall activity of the enzyme. Therefore, we have substituted the tryptophan in position 59 by tyrosine. This substitution led to an increase of enzymatic activity in comparison to variant RV to 425%. As the extent of this enhancement is unique so far we have crystallized and analyzed the structure of this variant in order to get more insights into the reasons for this. Here, we present the crystal structure of this so-called RNase T1-R2 at 2.1A resolution. The structure was determined by molecular replacement using the coordinates of the RV variant (PDB entry: 1Q9E). The data were refined to an R-factor of 18.7% and R(free) of 24%, respectively. The asymmetric unit contains three molecules and the crystal packing is very similar to that of variant RV.

Amino Acid Substitution↗

PubMed2005

An alanine in segment 3 of domain III (IIIS3) of the cockroach sodium channel contributes to the low pyrethroid sensitivity of an alternative splice variant.

In a previous study, we showed that two alternative exons (G1 and G2 encoding IIIS3-S4) were involved in the differential sensitivity of two cockroach sodium channel splice variants, BgNa(v)1-1 and BgNa(v)2-1 (previously called KD1 and KD2), to deltamethrin, a pyrethroid insecticide (Tan, et al., 2002b. Alternative splicing of an insect sodium channel gene generates pharmacologically distinct sodium channels. J. Neurosci. 22, 5300-5309.). Here, we report the identification of an amino acid residue in exon G2 that contributes to the low deltamethrin sensitivity of BgNa(v)2-1. Replacement of A1356 in BgNa(v)2-1 with the corresponding V1356 in BgNa(v)1-1 enhanced the sensitivity of the BgNa(v)2-1 channel to deltamethrin by six-fold. Conversely, substitution of V1356 with A1356 in BgNa(v)1-1 produced a recombinant BgNa(v)1-1 channel that was 5-fold more resistant to deltamethrin. These results demonstrate that A1356 contributes to the low sensitivity of BgNa(v)2-1 to deltamethrin. A1356V substitution also shifted the voltage-dependence of activation by 10 mV in the hyperpolarizing direction. Possible mechanisms by which this amino acid change affects the action of pyrethroids on the sodium channel are discussed.

Alanine↗

PubMed1989

Relation between infection with a subtype of HPV16 and cervical neoplasia.

A variant (HPV16b) of the previously described human papillomavirus (HPV)16 (now called HPV16a) has been identified by polymerase chain reaction assays. Compared with HPV16a, HPV16b has a 21bp deletion and several point mutations within the upstream regulatory region. All cervical carcinoma samples studied contained only HPV16a. HPV16b was found only rarely in cervical neoplasia but was common in the normal population.

Carcinoma↗

PubMed2002

CYP17 genetic polymorphism in endometrial cancer: are only steroids involved?

Initiation and/or promotion of endometrial cancer is known to be associated with estrogen and androgen (androstenedione) excess as well as with hyperinsulinemia/insulin resistance. It is possible that some allelic polymorphisms of the genes involved in steroidogenesis or steroid metabolism contribute to endometrial cancer susceptibility. We evaluated here the role of CYP17 biallelic (MspAI) polymorphism in 114 endometrial cancer patients compared with 182 healthy women. Our data demonstrated that A2/A2 CYP17 genotype, considered on the basis of initial breast cancer studies as 'unfavorable', was under-represented in endometrial cancer group (odds ratio 0.48, 95% confidence interval 0.25-0.89) that confirmed results of two other recent investigations. Carriers of this genotype were characterized by having lower blood insulin (by 120 min of oral glucose tolerance test 36.7+/-3.9 microU/ml vs. 90.4+/-16.7 microU/ml in postmenopausal women with A1/A1 genotype, P=0.04) and C-peptide levels (after night fasting 575.2+/-78.3 pg/ml vs. 978.9+/-115.7 pg/ml, respectively, P=0.04). No significant difference was found between the mean concentrations of testosterone, dehydroepiandrosterone sulfate and estradiol concentrations in patients-carriers of separate CYP17 genotypes. Thus, CYP17 polymorphism (namely, carrying the 'normal' A1/A1 genotype) might be one of the risk factors for endometrial cancer development. A1/A1 CYP17 variant may be associated with untraditional (non-steroidal) pathways that calls for corresponding preventive measures in high-risk groups.

Adult↗

PubMed1999

Rett syndrome and genetic drift.

An X chromosome gene is assumed to be responsible for the cause of Rett syndrome (RS). However, new genealogical observations suggest involvement of autosomal recessive gene(s) as well, at least in familial cases. To account for these and other recent observations, the theoretical model presented in 1990 by the authors of this paper is applied to the calculation of gene frequencies. Observed frequencies of sporadic and familial cases of RS are used, taking into account genetic drift in inbreeded areas. Moreover, an attempt is made to use the proportion of RS variants in familial and sporadic cases for the explanation of so called 'formes frustes', and as evidence for the existence of female as well as male carriers. The estimated frequency of the recessive autosome mutation, or possibly a frequent polymorphism, is 22.5%.

Alleles↗

PubMed1994

Mutations affecting the activity of toxic shock syndrome toxin-1.

Toxic shock syndrome toxin-1 (TSST-1), the potent staphylococcal exoprotein linked to most cases of the toxic shock syndrome, is a V beta-restricted T-cell mitogen (a so-called "superantigen"). TSST-ovine (TSST-O) is a natural variant of TSST-1, and is produced by certain ovine mastitis-associated strains of Staphylococcus aureus. Compared to TSST-1, TSST-O is only weakly mitogenic for leporine or murine splenocytes. It differs from TSST-1 at 7 amino acid residues over its 194 amino acid length. Terminus shuffling between the two proteins has suggested that their C-terminal differences (T69, Y80, E132, and I140 in TSST-1; 169, W80, K132, and T140 in TSST-O) are in part responsible for their discrepant mitogenic properties. In order to explore further the functional consequences of altering TSST-1 at residues 132 and 140, we engineered point mutants of TSST-1 at those positions. The mutant proteins were purified to homogeneity from culture supernants of a nontoxigenic strain of S. aureus using a combination of ultrafiltration, liquid-phase isoelectric focusing, and ion-exchange chromatography. The mutants retained global structural integrity as evidenced by circular dichroism spectroscopy, their preserved resistance to trypsin digestion, and their preserved binding to a neutralizing murine monoclonal antibody. The mutants were then tested for mitogenicity for human T-cells: The mutant I140T was approximately as active as wild-type TSST-1, while the mutant E132D was about 10-fold attenuated. On the other hand, the mutants E132A or E132K were each at least 1000-fold attenuated.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Toxins↗

PubMed1987

Accuracy of alternative representations for integrated biochemical systems.

The Michaelis-Menten formalism often provides appropriate representations of individual enzyme-catalyzed reactions in vitro but is not well suited for the mathematical analysis of complex biochemical networks. Mathematically tractable alternatives are the linear formalism and the power-law formalism. Within the power-law formalism there are alternative ways to represent biochemical processes, depending upon the degree to which fluxes and concentrations are aggregated. Two of the most relevant variants for dealing with biochemical pathways are treated in this paper. In one variant, aggregation leads to a rate law for each enzyme-catalyzed reaction, which is then represented by a power-law function. In the other, aggregation produces a composite rate law for either net rate of increase or net rate of decrease of each system constituent; the composite rate laws are then represented by a power-law function. The first variant is the mathematical basis for a method of biochemical analysis called metabolic control, the latter for biochemical systems theory. We compare the accuracy of the linear and of the two power-law representations for networks of biochemical reactions governed by Michaelis-Menten and Hill kinetics. Michaelis-Menten kinetics are always represented more accurately by power-law than by linear functions. Hill kinetics are in most cases best modeled by power-law functions, but in some cases linear functions are best. Aggregation into composite rate laws for net increase or net decrease of each system constituent almost always improves the accuracy of the power-law representation. The improvement in accuracy is one of several factors that contribute to the wide range of validity of this power-law representation. Other contributing factors that are discussed include the nonlinear character of the power-law formalism, homeostatic regulatory mechanisms in living systems, and simplification of rate laws by regulatory mechanisms in vivo.

Biochemistry↗

PubMed2002

Variant Creutzfeldt-Jakob disease: an unfolding epidemic of misfolded proteins.

Variant Creutzfeldt-Jakob disease (vCJD) is an emerging infectious disease believed to be the human manifestation of bovine spongiform encephalopathy (BSE). Variant CJD belongs to a family of human and animal diseases called transmissible spongiform encephalopathies (TSE). The pathogenesis of TSE is not fully understood, but a modified form of a normal cellular protein plays a central role. Current measures to control vCJD aim to prevent transmission of the infectious agent from animals to humans through food or pharmaceutical products and to prevent transmission from person to person via medical interventions. The anticipated development of preclinical diagnostic tests and treatments for vCJD will create new control options and difficult choices.

Creutzfeldt-Jakob Syndrome↗