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PubMed1996

Expression of human foamy virus reverse transcriptase involves a spliced pol mRNA.

In human foamy virus (HFV) the reverse transcriptase is expressed independently of the Gag protein as a 127-kDa Pol precursor molecule. Evaluating the mechanism of Pol expression we identified a spliced mRNA which uses the main 5' splice donor and a splice acceptor site located in the gag gene. The significance of this spliced transcript for HFV Pol expression was studied by constructing a virus with a mutated splice acceptor site. This virus was unable to express detectable Pol proteins after transient transfection. Replication of the mutant was studied by a sensitive assay based on HFV transactivator-stimulated expression of an integrated lacZ gene under control of the HFV long terminal repeat. Whereas in the first 2 weeks after transfection the mutant replicated 3 to 5 order of magnitude less well than wild-type virus, extracellular titers obtained thereafter were similar to those of wild-type virus. This increase in replication competence was accompanied by a reversion of the mutated splice acceptor site. The results underlined the importance of the spliced pol transcript for HFV replication and pointed to a second mechanism of Pol expression. Indicator gene assays suggest that this other mechanism is likely to be a transactivator-dependent cryptic promoter in the gag gene which gives rise to Pol-encoding transcripts.

Animals↗
PubMed2006

mAMSA resistant human topoisomerase IIbeta mutation G465D has reduced ATP hydrolysis activity.

Type II Human DNA Topoisomerases (topos II) play an essential role in DNA replication and transcription and are important targets for cancer chemotherapeutic drugs. Topoisomerase II causes transient double-strand breaks in DNA, forming a gate through which another double helix is passed, and acts as a DNA dependent ATPase. Mutations in topoII have been linked to atypical multi-drug resistance. Both human Topoisomerase II isoforms, alpha and beta, are targeted by amsacrine. We have used a forced molecular evolution approach to identify mutations conferring resistance to acridines. Here we report mutation betaG465D, which was selected with mAMSA and DACA and is cross-resistant to etoposide, ellipticine and doxorubicin. Resistance to mAMSA appears to decrease over time indicating a previously unreported resistance mechanism. G465D lies within the B' domain in the region that contacts the cleaved gate helix. There is a 3-fold decrease in ATP affinity and ATP hydrolysis and an altered requirement for magnesium in decatenation assays. The decatenation rate is decreased for the mutated G465D protein. And we report for the first time the use of fluorescence anisotropy with intact human topoisomerase II.

Acridines↗
PubMed1999

[Evaluation of a newly discovered LDL receptor mutation (exon 10, GAC>AAC, D271N, "FH Graz-1") in familial hypercholesterolemia-- a familystudy].

Heterozygous familial hypercholesterolemia (FH, prevalence 1:500) is a major cause of early atherosclerotic disease. Little is known about possible co-factors influencing individual patient's risk. We investigated this question in a large family carrying a new LDL-receptor-mutation. Genetic analysis of all exons of the LDL-receptor gene in the index case using polymerase chain reaction (PCR) and Denaturing Gradient Gel Electrophoresis (DGGE) revealed a previously unknown mutation in exon 10 (GAC > ACC, D471N, "FH Graz-1"). Investigation of 21 family members (15 females, 6 males), aged 17 to 86 years, revealed 9 female and 4 male carriers of the mutation. 7 female carriers aged 17 to 58 years show no clinical signs of macrovascular disease. An 86-year old female patient, who was asymptomatic until 85, recently suffered a transient cerebral ischemic attack. All these females were normotensive. The only hypertensive 76-year old patient (ex-smoker with a history of 15 pack years) suffers from angina pectoris. 2 male carriers of the mutation (32 and 38 years old) are asymptomatic. A 65-year old patient suffers from cardiovascular disease. A 49-year old patient had a coronary artery bypass graft after a myocardial infarction at the age of 37. Additionally he has a history of bilateral thrombendarterectomy of the carotid arteries and suffers from bilateral peripheral artery disease. This patient also carries the apoE-genotype 4/3, which might be responsible for his poor response to stain therapy, and needs extracorporal lipid elimination (LDL-C > 200 mg/dl under drug therapy). Both of his daughters are homozygous for the apoE-allele 3 and and responded well to stain therapy. Genetic analysis in patients with FH assures diagnosis, but is not sufficient to determine the individual patient's risk. A precise clinical examination remains the gold standard for individual risk evaluation.

Adolescent↗
PubMed1999

Phenotypic behavior of caveolin-3 mutations that cause autosomal dominant limb girdle muscular dystrophy (LGMD-1C). Retention of LGMD-1C caveolin-3 mutants within the golgi complex.

Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cell types (cardiac and skeletal). Autosomal dominant limb girdle muscular dystrophy (LGMD-1C) in humans is due to mutations within the caveolin-3 gene: (i) a 9-base pair microdeletion that removes three amino acids within the caveolin scaffolding domain (DeltaTFT) or (ii) a missense mutation within the membrane spanning domain (P --> L). The molecular mechanisms by which these two mutations cause muscular dystrophy remain unknown. Here, we investigate the phenotypic behavior of these caveolin-3 mutations using heterologous expression. Wild type caveolin-3 or caveolin-3 mutants were transiently expressed in NIH 3T3 cells. LGMD-1C mutants of caveolin-3 (DeltaTFT or P --> L) were primarily retained at the level of a perinuclear compartment that we identified as the Golgi complex in double-labeling experiments, while wild type caveolin-3 was efficiently targeted to the plasma membrane. In accordance with these observations, caveolin-3 mutants formed oligomers of a much larger size than wild type caveolin-3 and were excluded from caveolae-enriched membrane fractions as seen by sucrose density gradient centrifugation. In addition, these caveolin-3 mutants were expressed at significantly lower levels and had a dramatically shortened half-life of approximately 45-60 min. However, caveolin-3 mutants were palmitoylated to the same extent as wild type caveolin-3, indicating that targeting to the plasma membrane is not required for palmitoylation of caveolin-3. In conclusion, we show that LGMD-1C mutations lead to formation of unstable high molecular mass aggregates of caveolin-3 that are retained within the Golgi complex and are not targeted to the plasma membrane. Consistent with its autosomal dominant form of genetic transmission, we demonstrate that LGMD-1C mutants of caveolin-3 behave in a dominant-negative fashion, causing the retention of wild type caveolin-3 at the level of the Golgi. These data provide a molecular explanation for why caveolin-3 levels are down-regulated in patients with this form of limb girdle muscular dystrophy (LGMD-1C).

3T3 Cells↗
PubMed1995

An intron-containing meiosis-induced recombination gene, rec15, of Schizosaccharomyces pombe.

Mutations in the rec15 gene of Schizosaccharomyces pombe reduce meiotic recombinant frequencies, in the three intervals tested, as much as 1000-fold but have no detectable mitotic phenotype. The rec15 gene was mapped to within 1 cM of mat1. The gene was cloned by genetic complementation and its nucleotide sequence determined. Deletion analysis and gene replacement confirmed that the clones contained the rec15 gene on DNA fragments as short as 1.3 kb. The nucleotide sequence of the 1.3 kb fragment predicted that rec15 had a 49 bp intron separating two exons encoding a 180-amino-acid polypeptide product. This predicted intron was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. During thermally induced meiosis in a pat1-114 (Ts) mutant, the rec15 transcripts were induced to maximal levels at 2-3h but were present at much lower levels before and after this time. The transient induction of the transcripts and the phenotype of a rec15 null (deletion) mutation suggest that the rec15 gene product is required during the early stages of meiosis for meiotic recombination.

Amino Acid Sequence↗
PubMed1997

[The role of resistance to C active protein (R-APC) in a pediatric stroke].

INTRODUCTION: Activated protein C resistance is a recently identified thrombophylic state which results from a mutation in the factor V gene and has been shown to be an important risk factor for peripheral venous thrombosis. We report a case of paediatric stroke in whom we have identified APC resistance. CLINICAL CASE: A boy presented acutely at the age of 6 years with a severe right sided headache, vomiting, unsteadiness and drowsiness which worsened over a period of 40 hours. Prior to this episode, he was neurologically and developmentally normal except for occasional headaches. CT showed low attenuation in the left cerebellar hemisphere, and occipital lobe associated with acute hydrocephalus. Excision biopsy of the left cerebellar cortex revealed inflammation and possible infarction. Although he remained in a 'locked-in' state with a flaccid quadriparesis for six months, he improved and was left with a left side hemiplegia, multiple cranial nerve palsies and a visual field defect. He represented at the age of thirteen years with transient ischaemic attacks and was found be heterozygous for the factor V Leiden mutation. Since he has been warfarinised, his symptoms have improved. CONCLUSIONS: Although cerebellar stroke in childhood is rare, it has been underdiagnosed in the past. As recurrence is common, patients should be fully investigated and followed up long term. Screening for new factor such as APC-resistance is recommended.

Adolescent↗
PubMed2006

Activating mutations in the ABCC8 gene in neonatal diabetes mellitus.

BACKGROUND: The ATP-sensitive potassium (K(ATP)) channel, composed of the beta-cell proteins sulfonylurea receptor (SUR1) and inward-rectifying potassium channel subunit Kir6.2, is a key regulator of insulin release. It is inhibited by the binding of adenine nucleotides to subunit Kir6.2, which closes the channel, and activated by nucleotide binding or hydrolysis on SUR1, which opens the channel. The balance of these opposing actions determines the low open-channel probability, P(O), which controls the excitability of pancreatic beta cells. We hypothesized that activating mutations in ABCC8, which encodes SUR1, cause neonatal diabetes. METHODS: We screened the 39 exons of ABCC8 in 34 patients with permanent or transient neonatal diabetes of unknown origin. We assayed the electrophysiologic activity of mutant and wild-type K(ATP) channels. RESULTS: We identified seven missense mutations in nine patients. Four mutations were familial and showed vertical transmission with neonatal and adult-onset diabetes; the remaining mutations were not transmitted and not found in more than 300 patients without diabetes or with early-onset diabetes of similar genetic background. Mutant channels in intact cells and in physiologic concentrations of magnesium ATP had a markedly higher P(O) than did wild-type channels. These overactive channels remained sensitive to sulfonylurea, and treatment with sulfonylureas resulted in euglycemia. CONCLUSIONS: Dominant mutations in ABCC8 accounted for 12 percent of cases of neonatal diabetes in the study group. Diabetes results from a newly discovered mechanism whereby the basal magnesium-nucleotide-dependent stimulatory action of SUR1 on the Kir pore is elevated and blockade by sulfonylureas is preserved.

ATP-Binding Cassette Transporters↗
PubMed2004

Transient hyper-17-OHPnemia unrelated to cross-reactions with residual fetal adrenal cortex products.

OBJECTIVE: To clarify the pathogenesis of transient hyper-17alpha-hydroxyprogesteronemia, we initiated a laboratory investigation in a pre-term infant with persistently high serum 17alpha-hydroxyprogesterone (17-OHP) until 2 months of age. METHODS: Serum 17-OHP level was measured by high-performance liquid chromatography and radioimmunoassay, and gene analysis of CYP21A2 (21-hydroxylase) was performed. RESULT: Serum 17-OHP level on the 29th day of life was 25.4 ng/ml, and the urinary steroid profile showed low pregnanetriolone. Gene analysis of 21-hydroxylase disclosed no mutation, and 17-OHP normalized by 3 months of age without specific treatment. CONCLUSION: Transient elevations in 17-OHP, which do not appear related to cross-reactions with products of a residual fetal adrenal cortex, may occur in the first few months of life.

17-alpha-Hydroxyprogesterone↗
PubMed1995

Mammalian antioxidant protein complements alkylhydroperoxide reductase (ahpC) mutation in Escherichia coli.

The MER5 [now called the Aop1 (antioxidant protein 1) gene] was cloned as a transiently expressed gene of murine erythroleukaemia (MEL) cell differentiation and its antisense expression inhibited differentiation of MEL cells. We found that the Aop1 gene shows significant nucleotide sequence similarity to the gene coding for the C22 subunit of Salmonella typhimurium alkylhydroperoxide reductase, which is also found in other bacteria, suggesting it functions as an antioxidant protein. Expression of the Aop1 gene product in E. coli deficient in the C22-subunit gene rescued resistance of the bacteria to alkylhydroperoxide. The human and mouse Aop1 genes are highly conserved, and they mapped to the regions syntenic between mouse and human chromosomes. Sequence comparisons with recently cloned mammalian Aop1 homologues suggest that these genes consist of a family that is responsible for regulation of cellular proliferation, differentiation and antioxidant functions.

Animals↗
PubMed1979

Epidermal dystrophy. Occurrence after psoriasis therapy with psoralen and long-wave ultraviolet light.

Focal dystrophy of epidermal cells has been recognized in about half of 37 patients treated with psoralen and long-wave ultraviolet radiation (PUVA) for psoriasis. This lesion appeared in many patients by the end of the clearing phase of therapy and was present to a similar degree one year after the beginning of the treatment. It is probable that most such changes are transient, but the appearance suggests the possibility that cells have undergone somatic mutation that potentially could be dangerous.

Epidermis↗
PubMed2000

Regulation of the promoter for human immunoglobulin gamma3 germ-line transcription and its interaction with the 3'alpha enhancer.

The mechanism underlying the differential regulation of switching to human IgG subclasses is still largely unknown. We demonstrate that the region upstream of the initiation sites for gamma3 germ-line (GL) transcripts contains a functional promoter which is synergistically induced by IL-4, antibody to CD40 and phorbol dibutyrate in transient transfection assays in the human DG75 cell line. Linker-scanning mutations identified multiple elements in the 3' half of the evolutionarily conserved sequence that are required for inducibility. Electrophoretic mobility shift assays showed that Stat6 and NF-kappaB p50 / p65 are induced after stimulation, and bind to specific sequence motifs within the promoter. Overexpression of Stat6, NF-kappaB p50 / p65 and C / EBPgamma synergistically induced the GL gamma3 promoter. Insertion of DNA segments from the human 3' IgH regions, which may function as a locus control region for switch recombination, greatly activated the promoter in an orientation-independent manner. Duplication of the enhancer fragments resulted in a further increase of promoter activity. The greater enhancement of the HS1,2 fragment from the 3' alpha1 rather than the alpha2 locus may suggest a mechanistic explanation for the differential expression of various isotypes.

B-Lymphocytes↗
PubMed2001

Novel heterozygous missense mutation in the platelet glycoprotein Ib beta gene associated with isolated giant platelet disorder.

The glycoprotein (GP) Ib/IX/V complex plays an important role in primary hemostasis, serving as the platelet receptor for von Willebrand factor (vWF). Recent studies have shown that the phenotype caused by mutations in the subunits of the GPIb/IX complex spans a wide spectrum; from the normal phenotype, to isolated giant platelet disorders (GPD), and to the full-blown bleeding disorder, the Bernard-Soulier syndrome (BSS). We characterize here a novel missense mutation of the GPIb beta gene associated with isolated GPD. In the patient's platelets, the expression level of the GPIb/IX complex was moderately reduced compared with that of the GPIIb/IIIa complex, whereas the latter was expressed at higher levels than in a normal control. Immunoblot analysis showed normal electrophoretic mobility of GPIb alpha, GPIb beta, and GPIX. However, the amount of GPIb beta was approximately 66% of the normal value. DNA sequencing analysis revealed a novel heterozygous missense mutation in the GPIb beta gene that converts Arg (CGC) to Cys (TGC) at residue 17. Transient transfection studies demonstrated that mutant GPIb beta protein was not detected in transfected 293T cells. These findings indicated that null expression of the abnormal GPIb beta causes decreased expression of the complex and results in the GPD phenotype in the patient, and suggested that homozygosity of the mutation may lead to a BSS phenotype in vivo.

Adult↗
PubMed2006

Characterization of peroxide-bound heme species generated in the reaction of thermally tolerant cytochrome c552 with hydrogen peroxide.

Peroxide-bound heme species have been considered difficult to detect under physiological conditions because of their intrinsically transient properties. Cytochrome c552 (cyt c552), from Thermus thermophirus HB8, bearing a mutation to an alanine at Met69 (M69A) reacts with hydrogen peroxide (H(2)O(2)) to generate a detectable hydroperoxo-ferric heme ([Fe(3+)--OOH]) species at ambient temperature. EPR measurements during appropriate reaction periods reveal that the [Fe(3+)--OOH] species is in a preequilibrium state between the resting form of the cyt c552 variant and a subsequent intermediate, compound II with a protein radical. Addition of ascorbic acid to the reaction mixture of the cyt c552 variant and H(2)O(2) does not affect the formation of the [Fe(3+)--OOH] species,a result suggesting that the species is incompetent for the oxidation of even an oxidatively fragile substrate such as ascorbic acid. Another variant bearing an additional mutation to aspartic acid at Val49 (V49D/M69A) reveals that a highly hydrophobic heme cavity in cyt c552 accounts for the generation of the durable [Fe(3+)--OOH] species. The less polar environment inside the cavity is expected to prevent H(2)O from approaching the cavity. This would suppress protonation of the distal oxygen atom of the [Fe(3+)--OOH] species and retard subsequent dissociation of H(2)O from the OOH moiety.

Antioxidants↗
PubMed1986

Integrative transformation of Caenorhabditis elegans.

A technique for introducing exogenous DNA into the chromosomes of the nematode Caenorhabditis elegans is presented. A cloned C. elegans amber suppressor tRNA gene, sup-7, is used as a selectable marker. The activity of this amber suppressor is selected for by injecting worms which carry an amber termination mutation in a gene (tra-3) whose function is required for fertility. Transient expression of sup-7 is evidenced by the presence of fertile (rescued) animals in the generation after injection. In a fraction of cases, these fertile animals give rise to stable suppressor lines (eight have been characterized so far). Each of the stable suppressor lines carries injected DNA sequences. The suppressor activities have been mapped to chromosomal loci, indicating that the exogenous DNA has integrated into the genome. This technique has been used to introduce a chimeric gene containing a Drosophila heat shock promoter element fused to coding sequences from the Escherichia coli beta-galactosidase gene. This chimeric gene functions and is heat inducible in the resulting stably transformed lines.

Journal Article↗
PubMed2000

Doxorubicin represses CARP gene transcription through the generation of oxidative stress in neonatal rat cardiac myocytes: possible role of serine/threonine kinase-dependent pathways.

Doxorubicin (Dox), an anthracyclin antineoplastic agent, causes dilated cardiomyopathy. CARP has been identified as a nuclear protein whose mRNA levels are exquisitely sensitive to Dox. In this study we investigated the molecular mechanisms underlying the repression of CARP expression by Dox in cultured neonatal rat cardiac myocytes. Dox (1 micromol/l)-mediated decrease in CARP mRNA levels was strongly correlated with BNP but not with ANP mRNA levels. Hydrogen peroxide scavenger catalase (1 mg/ml) but not hydroxyl radical scavengers dimethylthiourea (10 mmol/l) or mannitol (10 mmol/l) blunted the Dox-mediated decrease in CARP and BNP expression. Superoxide dismutase inhibitor diethyldithiocarbamic acid (10 mmol/l), which inhibits the generation of hydrogen peroxide from superoxide metabolism, attenuated the repression. PD98059 (MEK1 inhibitor, 50 micromol/l), SB203580 (p38 MAP kinase inhibitor, 10 micromol/l), calphostin C (protein kinase C (PKC) inhibitor, 1 micromol/l), non-selective protein tyrosine kinase inhibitors genistein (50 micromol/l) or herbimycin A (1 micromol/l) failed to abrogate the downregulation of CARP and BNP expression by Dox. In contrast, H7 (30 micromol/l), a potent inhibitor of serine/threonine kinase, significantly blocked Dox-mediated downregulation of CARP and BNP expression. Transient transfection of a series of 5'-deletion and site-specific mutation constructs revealed that M-CAT element located at -37 of the human CARP promoter mediates Dox-induced repression of CARP promoter activity. These results suggest that a genetic response to Dox is mediated through the generation of hydrogen peroxide, which is selectively linked to the activation of H7-sensitive serine/threonine kinase distinct from PKC and well characterized mitogen-activated protein (MAP) kinases (ERK and p38MAP kinase). Furthermore, our data implicated M-CAT element as a Dox-response element within the CARP promoter in cardiac myocytes.

Animals↗
PubMed1997

Modulation of bovine papillomavirus DNA replication by phosphorylation of the viral E1 protein.

E1 is the DNA replication origin recognition protein for bovine papillomavirus (BPV), and it carries out enzymatic functions required for initiation of viral DNA replication. Cellular mechanisms likely play a role in regulating BPV DNA replication. We are investigating the role of phosphorylation of E1 on viral replication in vivo and on E1 activity in vitro. Serine 109 is a phosphoacceptor in vivo and is targeted by protein kinase A and protein kinase C in vitro. A viral genome carrying a serine 109 to alanine mutation replicates more efficiently than wild-type in vivo in a transient replication assay. Furthermore, purified mutant protein, while having wild-type levels of ATPase activity, is able to bind more origin-containing DNA than wild-type E1. Phosphorylation therefore appears to play a selective role in modulating a specific E1 function during viral DNA replication.

Adenosine Triphosphatases↗
PubMed2002

Photoreceptor degeneration and Ca2+ influx through light-activated channels of Drosophila.

We discuss in this chapter the role of Ca2+ homeostasis in maintaining the structural integrity of photoreceptor cells in Drosophila. Both insufficient and excessive amounts of Ca2+ in photoreceptor cells appear to lead to cell degeneration. Because one of the two classes of light-sensitive channels in Drosophila photoreceptors is highly Ca2+-permeable, how well this class of channels functions can profoundly affect Ca2+ homeostasis. We will begin by reviewing Drosophila phototransduction, emphasizing what is known about the mechanism of activation of light-sensitive channels. We will then describe Ca2+ entry through light-sensitive channels and the presumed mechanisms by which too little and too much Ca2+ entry can both cause photoreceptor degeneration. We will conclude the chapter with discussions of two examples of mutations known to cause unregulated Ca2+ entry through light-sensitive channels, leading to massive photoreceptor degeneration.

Animals↗
PubMed1991

Secretion of active truncated CD4 into Escherichia coli periplasm.

A truncated molecule containing the first 183 amino acid residues of the HIV-1 receptor, CD4, was made by periplasmic secretion in Escherichia coli. The signal sequence from the E. coli proteins OmpA, PhoA, or OmpF was fused to the truncated CD4, under the control of either the trp or the lac promoter. The processed material secreted into the periplasm reacted with monoclonal antibodies and exhibited binding activity to the HIV-1 envelope protein gp120. Not all of the processed product was recovered in the periplasm by osmotic shock, suggesting that either the material aggregated in the periplasm or, during secretion, the molecule assumed some transient conformation that interfered with its translocation across the inner membrane. A mutation in prlA (a gene involved in secretion) increased the level of processing, suggesting that secretion of a heterologous protein in E. coli can be optimized by manipulating the host secretion apparatus.

Amino Acid Sequence↗