Biomedical subjects

Richard Lathe

Publications and source records attributed to Richard Lathe.

10 recordsLinked to original sources

PubMed2006

Porphyrinuria in childhood autistic disorder: implications for environmental toxicity.

To address a possible environmental contribution to autism, we carried out a retrospective study on urinary porphyrin levels, a biomarker of environmental toxicity, in 269 children with neurodevelopmental and related disorders referred to a Paris clinic (2002-2004), including 106 with autistic disorder. Urinary porphyrin levels determined by high-performance liquid chromatography were compared between diagnostic groups including internal and external control groups. Coproporphyrin levels were elevated in children with autistic disorder relative to control groups. Elevation was maintained on normalization for age or to a control heme pathway metabolite (uroporphyrin) in the same samples. The elevation was significant (P < 0.001). Porphyrin levels were unchanged in Asperger's disorder, distinguishing it from autistic disorder. The atypical molecule precoproporphyrin, a specific indicator of heavy metal toxicity, was also elevated in autistic disorder (P < 0.001) but not significantly in Asperger's. A subgroup with autistic disorder was treated with oral dimercaptosuccinic acid (DMSA) with a view to heavy metal removal. Following DMSA there was a significant (P = 0.002) drop in urinary porphyrin excretion. These data implicate environmental toxicity in childhood autistic disorder.

Administration, Oral↗

PubMed2005

Early onset of puberty and early ovarian failure in CYP7B1 knockout mice.

CYP7B1 is the enzyme responsible for hydroxylation and termination of the estrogenic actions of the androgen metabolite, 5alpha-androstane-3beta, 17beta-diol (3betaAdiol). 3betaAdiol is estrogenic in ERalpha or ERbeta positive cells only if they do not express CYP7B1. In this study we show that female CYP7B1(-/-) mice experience early onset of growth of the uterus and mammary glands and commence estrus cycles 2 days earlier than their wild-type littermates. Adult mammary glands and uteri appear to be under continuous estrogenic stimulation. We conclude that, by cell-specific regulation of the estrogenicity of 3betaAdiol, CYP7B1 performs two major tasks: (i) it allows 3betaAdiol to have growth inhibitory effects through ERbeta and (ii) it permits estradiol-specific activation of estrogen receptors by protection of certain cells from the estrogenic effects of 3betaAdiol. When CYP7B1 is inactivated, 3betaAdiol activates estrogen receptors indiscriminately, and the overall effect is prolonged and inappropriate exposure to estrogen.

Androstane-3,17-diol↗

PubMed2005

Comparative proteomic analysis using samples obtained with laser microdissection and saturation dye labelling.

Comparative proteomic methods are rapidly being applied to many different biological systems including complex tissues. One pitfall of these methods is that in some cases, such as oncology and neuroscience, tissue complexity requires isolation of specific cell types and sample is limited. Laser microdissection (LMD) is commonly used for obtaining such samples for proteomic studies. We have combined LMD with sensitive thiol-reactive saturation dye labelling of protein samples and 2-D DIGE to identify protein changes in a test system, the isolated CA1 pyramidal neurone layer of a transgenic (Tg) rat carrying a human amyloid precursor protein transgene. Saturation dye labelling proved to be extremely sensitive with a spot map of over 5,000 proteins being readily produced from 5 mug total protein, with over 100 proteins being significantly altered at p < 0.0005. Of the proteins identified, all showed coherent changes associated with transgene expression. It was, however, difficult to identify significantly different proteins using PMF and MALDI-TOF on gels containing less than 500 mug total protein. The use of saturation dye labelling of limiting samples will therefore require the use of highly sensitive MS techniques to identify the significantly altered proteins isolated using methods such as LMD.

Alzheimer Disease↗

PubMed2003

Mono-allelic expression of variegating transgene locus in the mouse.

We have generated transgenic mice which express an ovine beta-lactoglobulin transgene during lactation. In two transgenic lines, BLG/7 and BLG/45, beta-lactoglobulin protein levels vary between siblings, reflected at the cellular level by a mosaic transgene expression pattern in the mammary tissue that is reminiscent of position effect variegation. To investigate whether this variegating expression profile can be affected by the introduction of an identical variegating locus on the homologous chromosome, we compared the beta-lactoglobulin expression profiles in mice hemizygous or homozygous for the transgene locus. In BLG/45 mice, milk protein analysis revealed that transgene expression was effectively doubled in homozygous compared to hemizygous mice. In contrast, beta-lactoglobulin protein in hemizygous and homozygous BLG/7 mice displayed a similar range; although minimum expression levels were doubled in the homozygous population, the maximum level of expression was indistinguishable between the two populations. Fluorescent in situ hybridisation (FISH) for transgene mRNA indicated that for a given protein level, the extent of cellular expression is similar in both BLG/7 populations. In homozygous mice genomic DNA and nuclear RNA FISH demonstrated that only one of the two BLG/7 loci is active in expressing cells, while two transcription foci were present in BLG/45 homozygous mice. This mono-allelic transgene expression pattern is not inherited through the germline, as hemizygous mice bred from homozygous parents expressed at the expected hemizygous population level. We discuss these observations in the context of known epigenetic events such as imprinting and trans-inactivation.

Alleles↗

PubMed2002

An endocrine pathway in the prostate, ERbeta, AR, 5alpha-androstane-3beta,17beta-diol, and CYP7B1, regulates prostate growth.

Epithelial proliferation of the ventral prostate in rodents peaks between 2 and 4 weeks of age, and by week 8, proliferating cells are rare. We have used ERbeta(-/-) and CYP7B1(-/-) mice to investigate the role of ERbeta and one of its ligands, 5alpha-androstane-3beta,17beta-diol (3betaAdiol), in growth of the ventral prostate. Before puberty, ERbeta was found in quiescent but not in proliferating cells, and proliferating cells occurred more frequently in ventral prostates of ERbeta(-/-) mice than in wild-type littermates. Treatment with 3betaAdiol decreased proliferation in wild-type but not in ERbeta(-/-) mice. In rats, treatment with 3betaAdiol from postnatal day 2 to 28 resulted in reduction in growth of ventral prostates. The prostates of CYP7B1(-/-) mice were hypoproliferative before puberty and smaller than those of their wild-type littermates after puberty. Because CYP7B1 represents the major pathway for inactivating 3betaAdiol in the prostate, we suggest that ERbeta, 3betaAdiol, and CYP7B1 are the components of a pathway that regulates growth of the rodent ventral prostate. In this pathway, ERbeta is an antiproliferative receptor, 3betaAdiol is an ERbeta ligand, and CYP7B1 is the enzyme that regulates ERbeta function by regulating the level of 3betaAdiol.

Androstane-3,17-diol↗

PubMed2002

Dehydroepiandrosterone (DHEA) metabolism in Saccharomyces cerevisiae expressing mammalian steroid hydroxylase CYP7B: Ayr1p and Fox2p display 17beta-hydroxysteroid dehydrogenase activity.

We have engineered recombinant yeast to perform stereospecific hydroxylation of dehydroepiandrosterone (DHEA). This mammalian pro-hormone promotes brain and immune function; hydroxylation at the 7alpha position by P450 CYP7B is the major pathway of metabolic activation. We have sought to activate DHEA via yeast expression of rat CYP7B enzyme. Saccharomyces cerevisiae was found to metabolize DHEA by 3beta-acetylation; this was abolished by mutation at atf2. DHEA was also toxic, blocking tryptophan (trp) uptake: prototrophic strains were DHEA-resistant. In TRP(+) atf2 strains DHEA was then converted to androstene-3beta,17beta-diol (A/enediol) by an endogenous 17beta-hydroxysteroid dehydrogenase (17betaHSD). Seven yeast polypeptides similar to human 17betaHSDs were identified: when expressed in yeast, only AYR1 (1-acyl dihydroxyacetone phosphate reductase) increased A/enediol accumulation, while the hydroxyacyl-CoA dehydrogenase Fox2p, highly homologous to human 17betaHSD4, oxidized A/enediol to DHEA. The presence of endogenous yeast enzymes metabolizing steroids may relate to fungal pathogenesis. Disruption of AYR1 eliminated reductive 17betaHSD activity, and expression of CYP7B on the combination background (atf2, ayr1, TRP(+)) permitted efficient (>98%) bioconversion of DHEA to 7alpha-hydroxyDHEA, a product of potential medical utility.

17-Hydroxysteroid Dehydrogenases↗

PubMed2002

Steroid and sterol 7-hydroxylation: ancient pathways.

B-ring hydroxylation is a major metabolic pathway for cholesterols and some steroids. In liver, 7 alpha-hydroxylation of cholesterols, mediated by CYP7A and CYP39A1, is the rate-limiting step of bile acid synthesis and metabolic elimination. In brain and other tissues, both sterols and some steroids including dehydroepiandrosterone (DHEA) are prominently 7 alpha-hydroxylated by CYP7B. The function of extra-hepatic steroid and sterol 7-hydroxylation is unknown. Nevertheless, 7-oxygenated cholesterols are potent regulators of cell proliferation and apoptosis; 7-oxygenated derivatives of DHEA, pregnenolone, and androstenediol can have major effects in the brain and in the immune system. The receptor targets involved remain obscure. It is argued that B-ring modification predated steroid evolution: non-enzymatic oxidation of membrane sterols primarily results in 7-oxygenation. Such molecules may have provided early growth and stress signals; a relic may be found in hydroxylation at the symmetrical 11-position of glucocorticoids. Early receptor targets probably included intracellular sterol sites, some modern steroids may continue to act at these targets. 7-Hydroxylation of DHEA may reflect conservation of an early signaling pathway.

Apoptosis↗

PubMed2002

Multiple effects of genetic background on variegated transgene expression in mice.

BLG/7 transgenic mice express an ovine beta-lactoglobulin transgene during lactation. Unusually, transgene expression levels in milk differ between siblings. This variable expression is due to variegated transgene expression in the mammary gland and is reminiscent of position-effect variegation. The BLG/7 line was created and maintained on a mixed CBA x C57BL/6 background. We have investigated the effect on transgene expression of backcrossing for 13 generations into these backgrounds. Variable transgene expression was observed in all populations examined, confirming that it is an inherent property of the transgene array at its site of integration. There were also strain-specific effects on transgene expression that appear to be independent of the inherent variegation. The transgene, compared to endogenous milk protein genes, is specifically susceptible to inbreeding depression. Outcrossing restored transgene expression levels to that of the parental population; thus suppression was not inherited. Finally, no generation-dependent decrease in mean expression levels was observed in the parental population. Thus, although the BLG/7 transgene is expressed in a variegated manner, there was no generation-associated accumulated silencing of transgene expression.

Animals↗

PubMed2002

Hybridization parameters revisited: solutions containing SDS.

Salt concentration governs nucleic acid hybridization according to the Schildkraut-Lifson equation. High concentrations of SDS are used in some common protocols, but the effects of SDS on hybridization stringency have not been reported. We investigated hybridization parameters in solutions containing SDS. With targets immobilized on nylon membranes and PCR- or transcription-generated probes, we report that the 50% dissociation temperature (Tm*) in the absence of SDS was 15 degrees C-17degrees C lower than the calculated Tm. SDS had only modest effects on Tm* [1% (w/v) equating to 8 mM NaCl]. RNA/DNA hybrids were approximately 11 degrees C more stable than DNA/DNA hybrids. Incomplete homology (69%) significantly reduced the Tm* for DNA/DNA hybrids (approximately /4degrees C; 0.45 degrees C/% nonhomology) but far less so for RNA/DNA hybrids (approximately 2.3 degrees C; approximately 0.07 degrees C/% non-homology); incomplete homology also markedly reduced the extent of hybridization. On these nylonfilters, SDS had a major effect on nonspecific binding. Buffers lacking SDS, or with low salt concentration, gave high hybridization backgrounds; buffers containing SDS, or high-salt buffers, gave reproducibly low backgrounds.

DNA Probes↗

PubMed2002

Targeted inactivation of the mouse locus encoding coagulation factor XIII-A: hemostatic abnormalities in mutant mice and characterization of the coagulation deficit.

Blood coagulation factor XIII (FXIII) promotes cross-linking of fibrin during blood coagulation; impaired clot stabilization in human genetic deficiency is associated with marked pathologies of major clinical impact, including bleeding symptoms and deficient wound healing. To investigate the role of FXIII we employed homologous recombination to generate a targeted deletion of the inferred exon 7 of the FXIII-A gene. FXIII transglutaminase activity in plasma was reduced to about 50% in mice heterozygous for the mutant allele, and was abolished in homozygous null mice. Plasma fibrin gamma-dimerization was also indetectable in the homozygous deficient animals, confirming the absence of activatable FXIII. Homozygous mutant mice were fertile, although reproduction was impaired. Bleeding episodes, hematothorax, hematoperitoneum and subcutaneous hemorrhage in mutant mice were associated with reduced survival. Arrest of tail-tip bleeding in FXIII-A deficient mice was markedly and significantly delayed; replacement of mutant mice with human plasma FXIII (Fibrogammin P) restored bleeding time to within the normal range. Thrombelastography (TEG) experiments demonstrated impaired clot stabilization in FXIII-A mutant mice, replacement with human FXIII led to dose-dependent TEG normalization. The mutant mice thus reiterate some key features of the human genetic disorder: they will be valuable in assessing the role of FXIII in other associated pathologies and the development of new therapies.

Animals↗