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S Hardy

Publications and source records attributed to S Hardy.

At least 55 records · Page 3Linked to original sources
PubMed1997

Construction of adenovirus vectors through Cre-lox recombination.

Two barriers prevent adenovirus-based vectors from having wide application. One is the difficulty of making new adenoviruses, and the second is the strong immunological reaction to viral proteins. Here we describe uses of Cre-lox recombination to overcome these problems. First, we demonstrate a simple method for constructing E1-substituted adenoviruses. Second, we demonstrate a method to construct adenovirus vectors carrying recombinant genes in place of all of the viral genes, so-called gutless adenovirus vectors. The pivotal feature in each method is the use of a negatively selected adenovirus named psi5. We engineered a cis-acting selection into psi5 by flanking its packaging site with loxP sites. When psi5 was grown in cells making a high level of Cre recombinase, the packaging site was deleted by recombination and the yield of psi5 was reduced to 5% of the wild-type level. To make a new E1-substituted virus, we used psi5 as a donor virus and recombined it with a shuttle vector via a loxP site. The resulting recombinant virus has a single loxP site next to the packaging site and therefore outgrows psi5 in the presence of Cre recombinase. To make a gutless virus, we used psi5 as a helper virus. The only viral sequences included in the gutless vector are those needed in cis for its replication and packaging. We found that a loxP site next to the packaging site of the gutless virus was necessary to neutralize homologous recombination between psi5 and the gutless viruses within their packaging domains.

Adenoviridae↗
PubMed1996

Sex difference in risk of torsade de pointes with d,l-sotalol.

BACKGROUND: The present study was undertaken to test the hypothesis that women are more prone than men to develop torsade de pointes (TdP) in a defined cohort of patients exposed to the QT-prolonging antiarrhythmic drug d,l-sotalol. METHODS AND RESULTS: In a database derived from 22 clinical trials involving 3135 adult patients who received oral d,l-sotalol (median follow-up, 164 days), TdP developed in 44 (1.9%) of 2336 men and in 33 (4.1%) of 799 women (P < .001). Logistic regression analysis identified female sex (P < .0001), presenting arrhythmia of sustained ventricular tachycardia or fibrillation (P < .0001), history of congestive heart failure (P < .001), and d,l-sotalol dose > 320 mg/d (P < .001) as factors most predictive of TdP; in addition to these, a serum creatinine > 1.4 mg/dL in women and > 1.6 mg/dL in men was weakly predictive (P < .05). After adjustment for these risk factors, women had threefold greater odds of developing TdP than men. The sex difference in TdP risk was age independent and could not be explained by differential dose-related bradycardic responses in women versus men. CONCLUSIONS: Women are at increased risk of developing TdP during-administration of d,l-sotalol. This finding needs to be taken into account, together with other TdP risk factors, when patients are treated with this antiarrhythmic agent. Given the consistency between the present and other recent observations, greater caution in women regarding use of QT-prolonging drugs, in general, is advisable.

Adult↗
PubMed1996

Casein kinase II increases the transcriptional activities of MRF4 and MyoD independently of their direct phosphorylation.

The myogenic regulatory factors (MRFs) are a subclass of a much larger group of basic helix-loop-helix transcription factors which includes members of the E protein such as E47, E2-2, and HEB. Although the MRFs are unique in their ability to confer a myogenic phenotype on nonmuscle cells, they require E protein partners to form a MRF-E protein heterodimer, which represents the functional myogenesis-inducing complex. The mechanisms controlling homodimer and heterodimer formation in vivo remain largely unknown, although it is likely that posttranslational modification of one or both basic helix-loop-helix partners is critical to this regulatory event. In this respect, MyoD and MRF4, both members of the MRF family, exist in vivo as phosphoproteins and contains multiple consensus phosphorylation sites, including sites for casein kinase II (CKII) phosphorylation. In this study, we demonstrate that overexpression of CKII increases the transcriptional activities of MRF4 and MyoD in vivo. Interestingly, mutation of the individual CKII sites within MRF4 and MyoF does not alter the ability of CKII to enhance MRF transcriptional activity, suggesting that the effect of CKII expression on the MRFs is indirect. Given that the MRFs require dimerization with E protein partners to activate muscle-specific transcription, the effects of CKII expression on E protein function also were examined. Our studies show that E47 serves as an in vitro substrate for CKII and that CKII-phosphorylated E-47 proteins no longer bind to DNA. These observations were confirmed by in vivo experiments showing that overexpressing of CKII produces a dramatic reduction in E47 homodimer-directed transcription. We conclude from these studies that CKII may act as a positive regulator of myogenesis by preventing E protein homodimers from binding to muscle gene regulatory elements.

Amino Acid Sequence↗
PubMed1995

The Xenopus laevis TM-4 gene encodes non-muscle and cardiac tropomyosin isoforms through alternative splicing.

A full-length cDNA clone coding for a 248-amino-acid tropomyosin (TM) was isolated from a Xenopus laevis (Xl) oocyte cDNA library. This TM is very similar to the members of the non-muscle (nm) TM family that includes rat TM-4, human TM30pl and chicken cardiac fibroblast FT-C. An RNase protection assay showed that the corresponding transcript is expressed ubiquitously and revealed the presence of an alternative transcript in adult heart RNA. A full-length cDNA clone was isolated from an adult heart cDNA library using a nm TM-encoding cDNA probe. It encodes a muscle TM homologous to chicken FT-C and the corresponding mRNA is expressed in adult RNA and to a very low level in adult skeletal muscle. The two cDNAs correspond to two alternatively spliced isoforms of TM generated from the Xl TM-4 gene. These data, together with published observations, demonstrated that, in this amphibian, the cardiac muscle TM are synthesized from the TM alpha and TM-4 genes.

Alternative Splicing↗
PubMed1995

The MLC1f/3f gene is an early marker of somitic muscle differentiation in Xenopus laevis embryo.

cDNAs clones encoding the MLC1f and MLC3f proteins of Xenopus laevis have been isolated from a stage 42 cDNA library. Sequence analysis reveals that the amphibian MLC1f and MLC3f isoforms are similar to the mammalian and avian cognates. The two isoforms share a common 141-amino-acid carboxy-terminal regions. These are 49 and 9 residues long for the MLC1f and MLC3f isoforms, respectively. This suggests a genomic organization similar to the mammalian and avian genes, with two promoters and alternative splicing. The developmental expression of the MLC1f/3f mRNAs was studied by Northern blot and RNase protection and their spatial expression analyzed by in situ hybridization. Both the MLC1f and MLC3f mRNAs can be detected in the developing embryo from the end of gastrulation and accumulate rapidly in the somitic mesoderm. Expression of the MLC1f/3f gene can also be detected in animal cap explants which have been induced to form mesodermal derivatives by exposure to activin A or bFGF. However, unlike other muscle-specific markers, neither transcript from the MLC1f/3f gene can be detected in embryonic or adult cardiac muscle, their expression being restricted to somitic muscle. Together, these data demonstrate that expression of the MLC1f/3f gene provides a sensitive and specific marker for skeletal muscle differentiation. Ectopic expression of myogenic factors in animal caps induces the expression of the MLC1f/3f gene, suggesting that the amphibian gene, like its mammalian and avian counterparts, is a regulatory target for members of the MyoD family of transcription factors.

Amino Acid Sequence↗
PubMed1995

Optic nerve decompression in cranial base fibrous dysplasia.

Fibrous dysplasia of the anterior cranial base involves the bony orbit and optic canal. Although fibrous dysplasia is benign, it may produce a mass effect along the course of the optic nerve, inducing visual disturbances. Optic canal decompression in patients without clinical signs of optic neuropathy is controversial. We describe five patients with extensive fibrous dysplasia of the anterior cranial base involving the orbit and optic canal. These patients underwent transcranial optic canal decompression before signs of severe visual loss during correction of dystopias and craniofacial deformity induced by fibrous dysplasia. Cranial orbital reconstruction was performed by means of split rib and cranial bone grafts. Postoperative follow-up did not reveal disturbances in visual function, extraocular motility, or evidence of cerebrospinal fluid fistulas. This suggests that early, radical resection of orbital fibrous dysplasia with optic canal decompression may be effective in preventing visual loss with minimal risk of other neurological sequelae. Subsequent orbital reconstruction involving split-thickness rib and cranial bone grafting yields satisfactory cosmetic results.

Adolescent↗
PubMed1995

"False" migration of rigid fixation appliances in pediatric craniofacial surgery.

Osseous fixation techniques have been widely used to provide rigid stabilization in the craniofacial skeleton. Reported sequelae of its usage has been limited to palpation of the screw-plate system and radiological imaging artifacts. Over the past 3 years we have identified miniplates, microplates, and wire sutures on the inner cranial table of the growing child. The observation of "false" migration of these appliances has provided the impetus to review these patients in more detail. Twenty patients underwent secondary cranial remodeling within a two-year period; 7 of these patients were seen to have "false" migration. There were no untoward sequelae in removal of these appliances, and no adverse neurological symptoms were seen.

Bone Plates↗
PubMed1994

Ethnic distribution of the glutathione transferase Mu 1-1 (GSTM1) null genotype in 1473 individuals and application to bladder cancer susceptibility.

Polycyclic aromatic hydrocarbons, found in cigarette smoke, food and industrial materials, are potential human carcinogens. Deficiency of detoxifying enzymes, such as glutathione transferases, may affect the metabolic fates of these chemicals and raise cancer risks in exposed individuals. The GSTM1 null genotype is a common form of glutathione transferase deficiency. Because knowledge of its ethnic distribution would be useful in epidemiologic studies, we measured the frequencies of the GSTM1 null genotype among healthy blacks, whites, Asian Indians, Chinese, Japanese, Koreans, Filipinos, Samoans and Hispanics. Rapid genotyping was done by use of a PCR assay, with dried blood spots on blotter paper as DNA templates. The frequency of the null genotype ranged from 0.31 among blacks to 0.88 among Samoans. The PCR assay was also applied to a pilot study of 114 bladder cancer cases from Kaiser Permanente Medical Center, Harbor City, California. DNA for these cases was obtained from paraffin-embedded surgical specimens. The overall odds ratio for bladder cancer with the GSTM1 null genotype was 1.4 (95% confidence interval 0.94-2.1), indicating no statistical difference in null genotype frequencies among bladder cancer patients compared to a healthy population. Large epidemiologic studies, which can be accomplished with dried blood spots or paraffin-embedded tissue specimens, may be useful for further assessment.

Adolescent↗
PubMed1994

Ethnic distribution of slow acetylator mutations in the polymorphic N-acetyltransferase (NAT2) gene.

The acetylation polymorphism may affect rates of activation or detoxification of common carcinogens, thereby influencing cancer risk. Our aim was to define the ethnic distribution of the major slow acetylator mutations in the polymorphic N-acetyltransferase gene, in order to provide background data for epidemiological studies. Our results contain new analyses on 803 individuals, including 365 new specimens and 438 specimens that had been partly characterized in an earlier study. Tests were done to establish the specificity and reproducibility (98%) of our PCR assays. The recognized slow acetylator mutations, 191A, 481T, 590A, and 857A (which correspond to alleles M4 and M4b; M1 and r3; M2/r2; and M3 and S3, respectively), accounted for nearly all slow acetylator alleles among blacks, whites, Asian Indians, Hispanics, Koreans, Japanese, Hong Kong Chinese, Taiwanese, Filipinos and Samoans. The ethnic distribution supports an interpretation that the acetylation polymorphism existed before Paleolithic splitting of human populations from Africa. We identified two additional NAT2 mutations, suggesting that other rare alleles are likely to be found.

Acetylation↗
PubMed1993

A new microcellular cytotoxicity test based on calcein AM release.

We present a microtest for cell-mediated immunity, based on the use of the Tarasaki tray and calcein AM vital dye. The number of target cells needed has been reduced to 500 per test with a corresponding tenfold reduction in the number of effector cells needed. Results were read at the rate of 1 second per test using a fluorimeter attached to a microscope. Each reaction was also confirmed visually with the use of ethidium bromide as a counterstain for dead cells. The calcein AM dye used to stain the living cells was shown to have a low spontaneous leakage rate--less than 15% in 4 hours at 37 degrees C. Dilutions of targets stained by calcein AM had a linear relationship with measured fluorescence values. NK cells, LAKs, and CTLs were readily detectable by this microtest. Quantitation of killing and kinetic analysis was readily performed with this test system. A significant positive correlation to 51Cr-release results was found. We conclude that the microtest should find wide application in studies of cell-mediated immunity.

Cell Line↗
PubMed1993

Fibroblast growth factor inhibits MRF4 activity independently of the phosphorylation status of a conserved threonine residue within the DNA-binding domain.

MRF4 is a member of the muscle-specific basic helix-loop-helix transcription factor family that also includes MyoD, myogenin, and Myf-5. Each of these proteins, when overexpressed in fibroblasts, converts the cells to differentiated muscle fibers that express several skeletal muscle genes, such as those for alpha-actin, muscle creatine kinase, and troponin I. Despite the fact that MRF4 functions as a positive transcriptional regulator, the MRF4 protein is subject to negative regulation by a variety of agents, most notably by exposure of cells to purified growth factors, such as basic fibroblast growth factor (bFGF). In an effort to establish whether bFGF inhibits MRF4 activity through specific posttranslational modifications, we examined whether MRF4 exists in vivo as a phosphoprotein and whether the phosphorylation status of the protein regulates its activity. Our results indicate that MRF4 is phosphorylated predominantly on serine residues, with weak phosphorylation occurring on threonine residues. Both cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) phosphorylate MRF4 in vitro as well as in vivo, and the overexpression of each kinase inhibits MRF4 activity and thus blocks terminal differentiation. PKC-directed phosphorylation of a conserved threonine residue (T-99) situated within the DNA-binding domain inhibits MRF4 from binding in vitro to specific DNA targets. However, although T-99 itself is essential for myogenic activity, our studies demonstrate that the phosphorylation status of T-99 does not play a major role in regulating MRF4 activity in vivo, since PKA, PKC, and bFGF inhibit the activity of MRF4 proteins in which the identified PKA and PKC sites have been mutated. We suggest that the negative regulation of MRF4 imposed by bFGF does not involve a direct modification of the protein at the identified PKA and PKC sites but instead may involve the modification of specific coregulators that interact with this muscle regulatory factor.

Amino Acid Sequence↗
PubMed1993

Slow acetylator mutations in the human polymorphic N-acetyltransferase gene in 786 Asians, blacks, Hispanics, and whites: application to metabolic epidemiology.

Our aim was to determine the population frequencies of the major slow acetylator alleles of the polymorphic N-acetyltransferase (NAT2) gene, whose locus maps to chromosome 8. We used allele-specific PCR amplification on 786 dried blood spots obtained from Hong Kong Chinese, U.S. Koreans, U.S. blacks, U.S. Hispanics, Germans, and U.S. whites. Our results show that four slow acetylator alleles can be detected as mutations at positions 481, 590, and 857 in the NAT2 gene. Recognized base substitutions at positions 341 and 803 need not be determined, because they were almost always associated with the 481T mutation. The known mutation at position 282 was strongly associated with the 590A mutation. The 481T, 590A, and 857A mutations accounted for virtually all of the slow acetylator alleles in Asian and white populations. The 857A mutation proved to be an Asiatic allele. The results will be useful in large-scale epidemiologic studies of cancer and other conditions potentially associated with the acetylator polymorphism.

Acetylation↗
PubMed1992

Isolation and characterization of cDNA clones encoding the skeletal and smooth muscle Xenopus laevis beta tropomyosin isoforms.

cDNAs clones corresponding to the skeletal and smooth muscle beta tropomyosins isoforms were isolated from a Xenopus laevis embryo cDNA library. Sequence analysis indicated that the two isoforms are coded by a single gene that uses two couples of alternative exons. The expression of the X. laevis beta tropomyosin gene closely resembles that of the mammalian gene but differs from the avian gene.

Amino Acid Sequence↗
PubMed1991

Characterization of muscle and non muscle Xenopus laevis tropomyosin mRNAs transcribed from the same gene. Developmental and tissue-specific expression.

We have isolated and characterized three Xenopus laevis tropomyosin cDNAs (XTM alpha 2, XTM alpha 4 and XTM05). XTM alpha 2 and XTM alpha 4 were isolated from a stage-42 embryo cDNA library and XTM05 from an oocyte cDNA library. XTM alpha 2 and XTM alpha 4 both code for a 284-amino-acid protein homologous to the skeletal alpha tropomyosin but diverge in their 3' untranslated sequences. This divergence is due to an absence of splicing in the early embryo. XTM05 codes for a 248-amino-acid non-muscle tropomyosin. Sequence analysis indicates that the three cDNAs are generated from a single gene which uses two promoters and possibly three sets of alternate exons. Northern blot analysis, with specific probes against each cDNA, was used to study the expression of the X. laevis alpha tropomyosin gene during development and in specific tissues. This analysis showed a different temporal usage of the two promoters and characterized the tissue-specific expression of the different transcripts.

Amino Acid Sequence↗
PubMed1991

Reinvestigation of DNA ligase I in axolotl and Pleurodeles development.

We have recently shown that the exclusion process causing the replacement of DNA ligases II by DNA ligase I in amphibian eggs after fertilization does not occur in the case of Xenopus laevis [Hardy, S., Aoufouchi, S., Thiebaud, P., and Prigent, C., (1991) Nucleic Acids Res. 19, 701-705]. Since this result is in contradiction with the situation reported in axolotl and Pleurodeles we decided to reinvestigate such results in both species. Three different approaches have been used: (1) the substrate specificity of DNA ligase I; (2) the DNA ligase-AMP adduct reaction and (3) the immunological detection using antibodies raised against the X.laevis DNA ligase I. Our results clearly demonstrate that DNA ligase I activity is associated with a single polypeptide which is present in oocyte, unfertilized egg and embryo of both amphibians. Therefore, the hypothesis of a change in DNA ligase forms, resulting from an expression of the DNA ligase I gene in axolotl and Pleurodeles early development must be rejected. We also show that, in contradiction with published data, the unfertilized sea urchin egg contains a DNA ligase activity able to join blunt ended DNA molecules.

Adenosine Monophosphate↗
PubMed1991

DNA ligase I from Xenopus laevis eggs.

We have purified the major DNA ligase from Xenopus laevis eggs and raised antibodies against it. Estimates from SDS PAGE indicate that this DNA ligase is a 180 kDa protein. This enzyme is similar to the mammalian type I DNA ligase which is presumed to be involved in DNA replication. We have also analysed DNA ligase activity during X. laevis early development. Unfertilized eggs contain the highest level of activity reflecting the requirement for a large amount of DNA replicative enzymes for the period of intense replication following fertilization. In contrast with previous studies on the amphibians axolotl and Pleurodeles, the major DNA ligase activity detected during X. laevis early development is catalysed by a single enzyme: DNA ligase I. And the presence of this DNA ligase I in Xenopus egg before fertilization clearly demonstrates that the exclusion process of two forms of DNA ligase does not occur during X. laevis early development.

Animals↗
PubMed1991

Development of a quality-of-movement measure for children with cerebral palsy.

Development of a suitable measure of quality of movement, or gross motor performance, for children with cerebral palsy is a complex undertaking. A variety of conceptual, methodological, and practical issues inherent in such a project are discussed in this article. We report on the methodology used in the planning and construction of the Gross Motor Performance Measure. The measure has been developed by a multicenter, interdisciplinary group of therapists, methodologists, research staff, and international experts. Five attributes of gross motor performance have been defined, scaled, and operationalized. Results of content validity studies demonstrate that the measure has adequate completeness, clarity, and potential for evaluating change in quality of movement in children who have cerebral palsy. The measure is currently undergoing extensive testing to determine the reliability, validity, and responsiveness of the obtained scores. [Boyce WF, Gowland C, Hardy S, et al. Development of a quality-of-movement measure for children with cerebral palsy.

Cerebral Palsy↗