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Biomedical subjects

E Richard

Publications and source records attributed to E Richard.

At least 37 records · Page 2Linked to original sources
PubMed1999

DNA damage and DNA damage-inducible protein Gadd45 following ischemia in the P7 neonatal rat.

Cerebral ischemia in adult rodents leads to the production of several types of lesions in the genomic DNA, followed by the activation of the damage-response indicator Gadd45. Our purpose was to investigate the structural changes that occur in chromatin DNA and repair processes after ischemic injury in neonatal brain. Neonatal ischemia was induced by the permanent left MCA occlusion in association with 1 h occlusion of the left common carotid artery in 7-day-old Wistar pups. Oligonucleosome fragments that are recognized as the characteristic DNA ladder was observed in a delayed fashion. Double-strand breaks result in high molecular weight fragments of 50- and 300-kbp as demonstrated by pulsed-field gel electrophoresis, and visualized by the TUNEL assay at 24 h of recovery. In contrast, DNA single-strand breaks, shown by the use of DNA polymerase I-mediated biotin-dATP nick translation were not so abundant. Gadd45 immunoreactivity was sequentially increased in vulnerable neurons in the infarct (4 to 24 h) and in sublethally injured neurons in the penumbra (24-48 h). Taken together, these findings suggest that Gadd45 responds to DNA damage following neonatal ischemia. Furthermore, repairing processes seem to be more active in the penumbra and therefore Gadd45 could have also a protective role in cerebral ischemia.

Animals↗
PubMed1999

Genetic heterogeneity in propionic acidemia patients with alpha-subunit defects. Identification of five novel mutations, one of them causing instability of the protein.

The inherited metabolic disease propionic acidemia (PA) can result from mutations in either of the genes PCCA or PCCB, which encode the alpha and beta subunits, respectively, of the mitochondrial enzyme propionyl CoA-carboxylase. In this work we have analyzed the molecular basis of PCCA gene defects, studying mRNA levels and identifying putative disease causing mutations. A total of 10 different mutations, none predominant, are present in a sample of 24 mutant alleles studied. Five novel mutations are reported here for the first time. A neutral polymorphism and a variant allele present in the general population were also detected. To examine the effect of a point mutation (M348K) involving a highly conserved residue, we have carried out in vitro expression of normal and mutant PCCA cDNA and analyzed the mitochondrial import and stability of the resulting proteins. Both wild-type and mutant proteins were imported into mitochondria and processed into the mature form with similar efficiency, but the mature mutant M348K protein decayed more rapidly than did the wild-type, indicating a reduced stability, which is probably the disease-causing mechanism.

Acid-Base Imbalance↗
PubMed1999

Overview of mutations in the PCCA and PCCB genes causing propionic acidemia.

Propionic acidemia is an inborn error of metabolism caused by a deficiency of propionyl-CoA carboxylase, a heteropolymeric mitochondrial enzyme involved in the catabolism of branched chain amino acids, odd-numbered chain length fatty acids, cholesterol, and other metabolites. The enzyme is composed of alpha and beta subunits which are encoded by the PCCA and PCCB genes, respectively. Mutations in both genes can cause propionic acidemia. The identification of the responsible gene, previous to mutation analysis, can be performed by complementation assay or, in some instances, can be deduced from peculiarities relevant to either gene, including obtaining normal enzyme activity in the parents of many patients with PCCB mutations, observing combined absence of alpha and beta subunits by Western blot of many PCCA patients, as well as conventional mRNA-minus result of Northern blots for either gene or beta subunit deficiency in PCCB patients. Mutations in both the PCCA and PCCB genes have been identified by sequencing either RT-PCR products or amplified exonic fragments, the latter specifically for the PCCB gene for which the genomic structure is available. To date, 24 mutations in the PCCA gene and 29 in the PCCB gene have been reported, most of them single base substitutions causing amino acid replacements and a variety of splicing defects. A greater heterogeneity is observed in the PCCA gene-no mutation is predominant in the populations studied-while for the PCCB gene, a limited number of mutations is responsible for the majority of the alleles characterized in both Caucasian and Oriental populations. These two populations show a different spectrum of mutations, only sharing some involving CpG dinucleotides, probably as recurrent mutational events. Future analysis of the mutations identified, of their functional effect and their clinical relevance, will reveal potential genotype-phenotype correlations for this clinically heterogeneous disorder.

Alternative Splicing↗
PubMed1998

An unusual late-onset case of propionic acidaemia: biochemical investigations, neuroradiological findings and mutation analysis.

UNLABELLED: We report a 5-year-old boy with propionic acidaemia who developed a rapidly fatal necrosis of the basal ganglia after an episode of clinical deterioration. Neither metabolic acidosis nor hyperammonaemia were present. Organic acid analysis in both urine and CSF showed increased levels of methylcitric and 3-hydroxypropionic acids. Propionic acidaemia was confirmed by demonstrating a propionyl-CoA carboxylase deficiency (11% of control value) in skin fibroblasts. DNA analysis revealed that the patient was a compound heterozygote for two mutations in the PCCB gene. CONCLUSION: Propionic acidaemia can present as a sudden and fatal neurological disease and not only as an organic aciduria with severe biochemical dis-turbances and progressive neurological deterioration.

Amino Acid Metabolism, Inborn Errors↗
PubMed1998

Human propionyl-CoA carboxylase beta subunit gene: exon-intron definition and mutation spectrum in Spanish and Latin American propionic acidemia patients.

Propionyl-CoA carboxylase (PCC) is a mitochondrial biotin-dependent enzyme composed of an equal number of alpha and beta subunits. Mutations in the PCCA (alpha subunit) or PCCB (beta subunit) gene can cause the inherited metabolic disease propionic acidemia (PA), which can be life threatening in the neonatal period. Lack of data on the genomic structure of PCCB has been a significant impediment to full characterization of PCCB mutant chromosomes. In this study, we describe the genomic organization of the coding sequence of the human PCCB gene and the characterization of mutations causing PA in a total of 29 unrelated patients-21 from Spain and 8 from Latin America. The implementation of long-distance PCR has allowed us to amplify the regions encompassing the exon/intron boundaries and all the exons. The gene consists of 15 exons of 57-183 bp in size. All splice sites are consistent with the gt/ag rule. The availability of the intron sequences flanking each exon has provided the basis for implementation of screening for mutations in the PCCB gene. A total of 56/58 mutant chromosomes studied have been defined, with a total of 16 different mutations detected. The mutation spectrum includes one insertion/deletion, two insertions, 10 missense mutations, one nonsense mutation, and two splicing defects. Thirteen of these mutations correspond to those not described yet in other populations. The mutation profile found in the chromosomes from the Latin American patients basically resembles that of the Spanish patients.

Amino Acid Substitution↗
PubMed1998

Identification and characterization of human cytochrome P450 isoforms interacting with pimozide.

Using human liver microsomes (HLMs) and recombinant human cytochrome P450 (CYP450) isoforms, we identified the major route of pimozide metabolism, the CYP450 isoforms involved, and documented the inhibitory effect of pimozide on CYP450 isoforms. Pimozide was predominantly N-dealkylated to 1,3-dihydro-1-(4-piperidinyl)-2H-benzimidazol-2-one (DHPBI). The formation rate of DHPBI showed biphasic kinetics in HLMs, which suggests the participation of at least two activities. These were characterized as high-affinity (K(m1) and Vmax1) and low-affinity (K(m2) and Vmax2) components. The ratio of Vmax1 (14 pmol/min/mg protein)/K(m1) (0.73 microM) was 5.2 times higher than the ratio of Vmax2 (244 pmol/min/mg protein)/K(m2) (34 microM). K(m2) was 91 times higher than K(m1). The formation rate of DHPBI from 25 microM pimozide in nine human livers correlated significantly with the catalytic activity of CYP3A (Spearman r = 0.79, P = .028), but not with other isoforms. Potent inhibition of DHPBI formation from 10 microM pimozide was observed with ketoconazole (88%), troleandomycin (79%), furafylline (48%) and a combination of furafylline and ketoconazole (96%). Recombinant human CYP3A4 catalyzed DHPBI formation from 10 microM pimozide at the highest rate (V = 2.2 +/- 0.89 pmol/min/pmol P450) followed by CYP1A2 (V = 0.23 +/- 0.08 pmol/min/pmol P450), but other isoforms tested did not. The K(m) values derived with recombinant CYP3A4 and CYP1A2 were 5.7 microM and 36.1 microM, respectively. Pimozide itself was a potent inhibitor of CYP2D6 in HLMs when preincubated for 15 min (Ki = 0.75 +/- 0.98 microM) and a moderate inhibitor of CYP3A (Ki = 76.7 +/- 34.5 microM), with no significant effect on other isoforms tested. Our results suggest that pimozide metabolism is catalyzed mainly by CYP3A, but CYP1A2 also contributes. Pimozide metabolism is likely to be subject to interindividual variability in CYP3A and CYP1A2 expression and to drug interactions involving these isoforms. Pimozide itself may inhibit the metabolism of drugs that are substrates of CYP2D6.

Antipsychotic Agents↗
PubMed1997

Three novel splice mutations in the PCCA gene causing identical exon skipping in propionic acidemia patients.

Propionyl-CoA carboxylase (PCC) is a mitochondrial, biotin-dependent enzyme involved in the catabolism of branched chain amino acids, odd chain fatty acids, and other metabolites. PCC consists of non-identical subunits, alpha and beta, encoded by the PCCA and PCCB genes, respectively. Inherited deficiency of PCC due to mutations in either the PCCA or the PCCB gene results in propionic acidemia (PA), a clinically heterogeneous disorder with a severe, often lethal, neonatal form, and a mild, later onset form. To characterize PCCA gene mutations responsible for PCC deficiency, we analyzed RT-PCR products obtained from cultured fibroblasts from Spanish PCC-alpha deficient patients. In three patients, smaller than normal PCR products were observed, and sequence analysis revealed the deletion of a 54-bp exon in the cDNA. Sequencing of genomic DNA from these three patients led to the identification of three novel mutations in the PCCA gene, two short deletions and one small insertion, adjacent to short direct repeats, and all of them affecting the consensus splice sites of the skipped exon. These mutations, 1771IVS-2del9, 1824IVS+3del4, and 1824IVS+3insCT, are the cause of the aberrant splicing of the PCCA pre-mRNA and result in an in-frame deletion of 54 nucleotides in the cDNA, probably leading to an unstable protein structure which is responsible for the lack of activity leading to PCC deficiency in these patients.

Amino Acid Metabolism, Inborn Errors↗
PubMed1997

[Munchausen syndrome by proxy].

Munchausen syndrome by proxy is a form of child abuse presenting as a disease produced or simulated by a parent, the mother in most cases. Its diagnosis is uneasy because of its miscellaneous and unusual clinical presentation and of the misleading apparently normal attitude of the parents. Physicians may participate in the abuse by insistently looking for diagnostic and therapeutic measures, therefore contributing to the significant mortality of the syndrome. It is therefore important that physicians consider Munchausen syndrome in any ambiguous situation in order to protect the child by an early diagnosis.

Child↗
PubMed1994

Effect of a conjugated oestrogen (Premarin) cream on ageing facial skin. A comparative study with a placebo cream.

The effects of Premarin cream on ageing facial skin were studied in a randomised, double-blind, parallel group study. Fifty-four women aged 52-70 years who had moderate to severe facial cutaneous ageing, applied 1 g of either Premarin cream (0.625 mg conjugated oestrogens per gram of cream), or placebo cream (same composition with the exclusion of conjugated oestrogens) to the face nightly for 24 weeks. Each morning these women protected their face with a sunblock SPF 15. Skin thickness was measured by B-scan ultrasonic echography, and skin microrelief by profilometry. Each subject's facial appearance was also evaluated by the subject herself and by the clinician. A statistically significant difference (P = 0.013) in favour of Premarin cream was detected in skin thickness at week 24. Skin thickness (dermal plus epidermal) for the women who used Premarin cream increased from 1.56 +/- 0.20 mm at baseline to 1.68 +/- 0.19 mm, compared with 1.52 +/- 0.20 mm at baseline to 1.59 +/- 0.19 mm in the placebo group. Premarin cream was also significantly more effective than placebo cream in improving fine wrinkles according to the results at weeks 12 and 24 (P = 0.010 and P = 0.012, respectively). Significant improvement from baseline was detected in both groups for roughness, laxity and mottled pigmentation and/or lentigines; however, there was no significant difference in these parameters between the two treatment groups. No subjects discontinued treatment for a safety reason. In conclusion, Premarin cream produced better results than the placebo cream; the difference was statistically significant for skin thickness and fine wrinkles. Premarin cream was well tolerated locally, and its general safety was good.

Administration, Topical↗
PubMed1989

In-vivo transperitoneal fertilization.

We present the technique of in-vivo transperitoneal fertilization (IVTPF) as a first approach to infertility treatment in couples with male subfertility or unexplained factors. The technique is statistically less successful but also less invasive than either gamete intra-Fallopian transfer (GIFT) or in-vitro fertilization - embryo transfer (IVFET) and offers considerable advantages over intrauterine insemination (IUI). The IVTPF technique involves transperitoneal transfer of processed spermatozoa within the pouch of Douglas after induction of ovulation. We report our 4-year experience with IVTPF which includes 136 treatment cycles in 89 couples. Eight pregnancies were achieved in 89 patients (9%) and 136 treatment cycles (7%). Fifty-one patients (57%) received IVTPF for only one treatment cycle; seven of the eight IVTPF pregnancies occurred in this group. An ectopic pregnancy resulted in one of the eight IVTPF pregnancies (13%). The functional quality of the sperm in those couples who achieved pregnancy was statistically superior to those couples who did not conceive. However, pregnancy was also obtained in case of severe oligozoospermia. Based on our experience, we feel IVTPF to be a very reasonable first approach in patients with no pelvic pathology and with infertility secondary to male factors or unexplained causes.

Adult↗
PubMed1988

Complications of the Camey procedure.

Since the authors' first case in 1958, 221 bladder replacements in more than 500 radical cystectomies have been performed in their department. They discuss perioperative complications; postoperative complications including mortality, gastrointestinal problems, urologic problems, and infections; and late complications such as ileal bladder problems, upper urinary tract problems, secondary diversions, and incontinence.

Humans↗
PubMed1986

Prevalence of nurse smokers and variables identified with successful and unsuccessful smoking cessation.

A mailed survey of 823 randomly selected registered nurses, constituting an 82% return rate, was conducted to determine the prevalence and quit rate of smoking related behaviors. Variables identified with successful quitters and continuing smokers also were investigated. The findings revealed that nurses have a decreased proportion of current smokers, an increased proportion of never smokers, and an increased quit smoking rate. The smoking cessation method used most frequently by successful quitters was "cold turkey".

Adult↗