Evaluation of Reference Genes for Quantitative PCR in Four Tissues from Rabbits with Hypercholesterolaemia

Abstract Rabbit with hypercholesterolaemia is an important model for studying cholesterol metabolism disease. This study aimed to evaluate the expression stability of nine reference genes for quantitative PCR (qPCR) analysis in adrenal gland, liver, spleen, and kidney tissue from rabbits with hypercholesterolaemia. In total, 30 male Harbin Large White (HLW) rabbits were fed a normal feed (n = 15) or a high cholesterol feed (n = 15) for 8 weeks to induce hypercholesterolaemia. Nine reference genes were verified by qPCR using cDNA extracted from rabbit tissue samples. For qPCR analysis, reference genes were evaluated using the RefFinder and GeNorm algorithms. Overall, seven rabbits with hypercholesterolaemia were identified based on body weight and total cholesterol measurements. Combining the results of the RefFinder and GeNorm algorithms, the most stable reference genes were hypoxanthine phosphoribosyltransferase 1 (Hprt1) and eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) in the adrenal gland, β-2-microglobulin (B2m) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) in the liver, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz) and Gapdh in the spleen, and peptidylprolyl isomerase (Ppia), β-actin (Actb), succinate dehydrogenase complex subunit A flavoprotein (Sdha), and B2m in the kidney. Taken together, our results confirmed that Hprt1 and Eef1a1, B2m and Gapdh, Ywhaz and Gapdh, and Ppia, Actb, Sdha, and B2m were the best reference genes for qPCR analyses in adrenal gland, liver, spleen, and kidney tissue, respectively, of rabbits with hypercholesterolaemia.

Validation of Housekeeping Genes for Gene Expression Analysis in Glioblastoma Using Quantitative Real-Time Polymerase Chain Reaction

BACKGROUND: Quantitative real-time polymerase chain reaction (qPCR) is the most reliable tool for gene expression studies. Selection of housekeeping genes (HKGs) that are having most stable expression is critical to carry out accurate gene expression profiling. There is no 'universal' HKG having stable expression in all kinds of tissues under all experimental conditions. METHODS: The present study aims to identify most appropriate HKGs for gene expression analysis in glioblastoma (GBM) samples. Based on literature survey, six most commonly used HKGs that are invariant in GBM were chosen. We performed qPCR using RNA from formalin fixed paraffin embedded GBM samples and normal brain samples to investigate the expression pattern of HPRT, GAPDH, TBP, B2M, B2M, RPL13A, and RN18S1 with different abundance. A simple Deltacycle threshold approach was employed to calculate the fold change. RESULTS: Our study shows that the expression of RPL13A and TBP were found to be most stable across all the samples and are thus suitable for gene expression analysis in human GBM. Except for TBP, none of the other conventionally used HKGs in GBM studies e.g., HPRT and GAPDH were found to be suitable as they showed variation in RNA expression. CONCLUSION: Validation of HKGs is therefore immensely specific for a particular experimental setup and is crucial in assessing any new setup.

A Case of Lesch-Nyhan Syndrome Manifesting Only Chronic Gouty Arthritis without Neurologic Symptom

Deficiency of hypoxanthine-guanine phosphoribosyltransferase is a purine nucleotide disorder and is the most common genetic cause of uric acid overproduction. This disease has a wide range of spectrum with regard to neurological features depending on the extent of the enzymatic deficiency. Complete deficiency of hypoxanthine-guanine phosphoribosyltransferase, called Lesch-Nyhan syndrome, is presented with hyperuricemia and characteristic neurological manifestation and self-mutilation. Partial hypoxanthine-guanine phosphoribosyltransferase--deficient patients are presented with a various intensities of the aforementioned symptoms, from almost normal neurologic manifestation to a severe form along with hyperuricemia. We report a twenty-year-old man with complete hypoxanthine-guanine phosphoribosyltransferase mutation and Lesch-Nyhan sydrome, who manifested gouty arthritis without neurologic symptom.

A Case of Lesch-Nyhan Disease Manifesting Gouty Arthritis without Self-mutilation / 대한신장학회지

Lesch-Nyhan disease is a very rare X-linked recessive disorder characterized by mental retardation, spasticity resembling cerebral palsy, choreoathetosis, self-mutilation and hyperuricemia. Self-mutilative behavior is a hallmark of the disease. The underlying defect is a deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). We report on a fourteen-year-old boy, who manifested gouty arthritis and mild renal insufficiency with Lesch-Nyhan disease, lacking self-mutilative behavior in spite of undetectable HPRT activity. Though there were several reports about some cases of Lesch-Nyhan disease in the past Korean literature, the cases were classic forms with definite neurological manifestation. As far as we know, this is the first case of Lesch-Nyhan disease without self-mutilation in Korea.

Determinación de las constantes cinéticas de la enzima hipoxantina - guanina fosforribosiltransferasa (HGPRT) en controles normales y en una familia afectada por el Sindrome de Lesch-Nyhan / Determination of the Kinetic constants of hypoxanthine guanine phosphoribosyl transférase enzyme (HGPRT) in normal controls and in a family affected by Lesch-Nyhan’s syndrome

El sindrome de Lesch-Nyhan (SLN) es producido por una deficiencia total de la enzima Hipoxantina-Guanina fosforribosiltransferasa (HGPRT). Las madres de niños afectados por el SLN son heterocigotas obligadas ya que la enfermedad se hereda de forma recesiva ligada al cromosoma X. Una de las células utilizadas para determinar la enfermedad es el eritrocito; sin embargo, la determinación de la condición portadora de las madres con niños afectados no se puede hacer en estas células ya que presentan una actividad que cae dentro del rango considerado normal.Esto sucede porque el eritrocito deficiente en la enzima HGPRT es destruido antes de que alcance la circulación sanguínea. Aplicando los principios cinéticos de Lineweaver-Burk y mediante espectrofotometría se determino la cinética de la (HGPRT) extraída de los eritrocitos de una familia que sufre el Sindrome de Lesch-Nyhan y se compararon con la cinética de esta enzima utilizando eritrocitos de 10 individuos sanos y normales procesados de igual forma. Los dos sustratos estudiados fueron la Guanina y el Fosforribosilpirofosfato (PRPP). La Guanina en individuos normales presentó un rango de Vmax entre 1.7 a 2.8 μmol de GMP/ min/ g Hb. mientras que el rango presentado para la Km fue de 10 a 25 μM. El PRPP en los controles normales presentó un rango para la Vmax de 2 a 2.7 μmol de GMP/min/g Hb y el rango para la Km fue de 301 a 590 μM. estos valores corresponden a lo reportado por otros autores. En la familia estudiada la cual tiene dos niños que padecen el síndrome de Lesch- Nyhan, tanto el padre como la hija presentaron cinéticas que caen dentro del rango considerado normal, mientras que la madre presentó una alteración en la Km para el PRPP ( 1176 μ M ). Cuando se comparó la regresión linear de las pendientes presentadas por los controles con la presentada por la paciente portadora de la enfermedad se encontró que estadísticamente son muy diferentes, diferencia que es ocasionada por el valor elevado de la Km de esta paciente por el sustrato PRPP.

The syndrome of Lesch-Nyhan (SLN) is produced by total deficiency of the enzyme Hipoxanthine-Guanine Phosphoribosyltransferase (HGPRT). The mothers of children affected by the SLN are obliged heterocigotes since the illness is inherited from a recessive way bound to the chromosome X. One of the utilized cells to diagnostic the illness is the erythrocyte; however, the determination of the condition carrier of the mothers with affected children cannot make in these cells since they present an activity that falls inside the normal considered range. This happens because the faulty erythrocyte in the enzyme HGPRT is destroyed before it reaches the blood stream. Applying the kinetic principles of Lineweaver-Burk and by means of espectrophotometry I to carry out the kinetic of the HGPRT enzyme extracted of the erythrocytes of a family that it suffers the SLN and they were compared with the kinetics of this enzyme in erythrocytes of ten individuals health and normal. The two studied substrates were the Guanine and the phosphorybosylpirophosphate(PRPP). The Guanine in normal individuals presented a range of Vmax among 1.7 at 2.8 μmol GMP/min/gHb, while the range presented for the Km went from 10 to 25 μ M. The PRPP in the normal controls presented a range for the Vmax from 2 at 2.7μmol GMP/min/gHb and the range for the Km went from 301 to 590 μM. These ranges are similar to those reported by other authors. In the estudied family which has two children that suffer the SLN , as much the father as the daughter presented kinetic that fall inside the normal considered range, while the mother presented increased the km for the PRPP ( the value of Km for the PRPP of the mother of the children affected by the syndrome was of 1176 μ M. When we compared the pendent the normal controls with the obliged heterocigotes pendent for the PRPP substrate was statisticaly different owing to high Km value.

Partial HPRT deficiency (Kelley-Seegmiller syndrome).

Hypoxanthine-guanine phosphoribosyl transferase (HPRT) deficiency is an X-linked defect of purine metabolism. Clinical manifestations are usually related to the degree of enzyme deficiency; complete HPRT deficiency (Lesh-Nyhan Syndrome) presenting with severe neurological or renal symptoms, or partial HPRT deficiency (Kelley-Seegmiller syndrome) manifesting as a gout-urolithiasis syndrome. We report a case of partial HPRT deficiency presenting as chronic tophaceous gout, mental retardation, nephrolithiasis and family history suggestive of X-linked inheritance, for its rarity.

Low doses of gamma ionizing radiation increase hprt mutant frequencies of TK6 cells without triggering the mutator phenotype pathway

The TK6 lymphoblastoid cell line is known to be mismatch repair (MMR) and p53 proficient. Deficiency in MMR results in a mutator phenotype characterized by microsatellite instability (MSI) and increased hprt mutant frequency (MF). Increased hprt MF is also a biomarker of effect for exposure to ionizing radiation. In order to test if a mutator phenotype could be induced by low doses of gamma ionizing radiation, an hprt cloning assay and a MSI investigation were performed after radiation exposure. The spontaneous MF was 1.6 x 10-6. The groups exposed to 0.2, 0.5 and 1.0 Gy had hprt MFs of 2.3, 3.3 and 2.2 x 10-6, respectively. The spontaneous MSI frequency per allele in non-selected cells was 5.4 x 10-3, as evidenced at the loci D11S35, nm23-H1, D8S135 and p53. MSI frequencies in the groups exposed to 0.2, 0.5 and 1.0 Gy were found to be < 4.7, < 7.7 and < 12 x 10-3, respectively. The frequencies of hprt mutants and MSI found in this study suggest that low doses of ionizing radiation increase hprt mutant frequency without triggering the mutator phenotype pathway.

Apoptosis-Induced Gene Profiles of a Myeloma Cell P3-X63-Ag8.653

BACKGROUND: Apoptosis is a physiologic phenomenon involved in development, elimination of damaged cells, and maintenance of cell homeostasis. Deregulation of apoptosis may cause diseases, such as cancers, immune diseases, and neurodegenerative disorders. The mouse myeloma cell P3-X63-Ag8.653 (v653) is an HGPRT deficient (HGPRT) mutant strain. High dependency on de novo transcription and translation of aminopterin induced apoptosis of this cell seems to be an ideal experimental system for searching apoptosis-induced genes. METHODS & RESULTS: For searching apoptosis-related genes we carried out GE-array (dot blot), Affymetrix GeneChip analysis, Northern analysis and differential display-PCR techniques. The chip data were analyzed with three different programs. 66 genes were selected through Affymetrix GeneChip analyses. All genes selected were classified into 8 groups according to their known functions. They were Genes of 1) Cell growth/maintenance/death/ enzyme, 2) Cell cycle, 3) Chaperone, 4) Cancer/disease-related genes, 5) Mitochondria, 6) Membrane protein/signal transduction, 7) Nuclear protein/nucleic acid binding/ transcription binding and 8) Translation factor. Among these groups number of genes were the largest in the genes of cell growth/maintenance/death/enzyme. Expression signals of most of all groups were peaked at 3 hour of apoptosis except genes of Nuclear protein/nucleic acid binding/ transcription factor which showed maximum signal at 1 hour. CONCLUSION: This study showed induction of wide range of proapoptotic factors which accelerate cell death at various stage of cell death. In addition apoptosis studied in this research can be classified as a type 2 which involves cytochrome c and caspase 9 especially in early stages of death. But It also has progressed to type 1 in late stage of the death process.

Three Cases of Lesch-Nyhan Syndrome: Cases report

Lesch-Nyhan syndrome is a rare X-linked recessive metabolic disorder characterized by developmental delay, hyperuricemia, choreoathetosis, spasticity, mental retardation, and compulsive self-injurious behavior. This disorder results from a complete deficiency of the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGPRT). This syndrome is often misdiagnosed to cerebral palsy and clinical manifestations are usually related to the degree of enzyme deficiency. Complete HGPRT deficiency presents with severe specific neurologic manifestation and nephrolithiasis leading to fatal kidney damage. This report highlighted the importance of clinical awareness leading to early diagnosis and therapy for prevention of the self mutilation and renal failure, even if we couldn't inhibit the progression of neuro-psychotic symptoms.

Investigating genetic damage of workers occupationally exposed to methotrexate / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study genetic damage of workers alone occupationally exposed to methotrexate (MTX) with three end-points.</p><p><b>METHODS</b>The blood samples from 21 workers exposed to MTX and 21 controls were detected with micronucleus test, comet assay, hprt gene mutation test and TCR gene mutation test.</p><p><b>RESULTS</b>The mean micronuclei rate (MNR) and mean micronucleated cells rate (MCR) in 21 workers were 10.10 per thousand +/- 0.95 per thousand and 8.05 per thousand +/- 0.75 per thousand, respectively, which were significantly higher than those (5.48 per thousand +/- 0.82 per thousand and 4.38 per thousand +/- 0.58 per thousand) in control (P < 0.01). The mean tail length (MTL) of 21 workers and 21 controls were (1.30 +/- 0.06) microm and (0.07 +/- 0.01) microm, respectively, there was significant difference between workers and controls (P < 0.01). But the difference between workers and controls for mean tail moment (MTM) was not significant (P > 0.05). The average mutation frequency (Mf-hprt) of hprt and (Mf-TCR) of TCR in workers were 1.00 per thousand +/- 0.02 per thousand and (6.87 +/- 0.52) x 10(-4), respectively, which were significantly higher than those [0.86 per thousand +/- 0.01 per thousand and (1.67 +/- 0.14) x 10(-4)] in control (P < 0.01).</p><p><b>CONCLUSION</b>The genetic damage to some extent appeared in workers occupationally exposed to methotrexate.</p>

Molecular analysis of hprt mutation in B6C3F1 mice exposed to ozone alone and combined treatment of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone and/or dibutyl phthalate for 32 and 52 weeks

Potential toxicological interactions of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and/or dibuthyl phthalate (DBP) on ozone were investigated after 32- and 52-wk exposures using hprt mutation assay. Male and female B6C3F1 mice exposed to ozone (0.5 ppm), NNK (1.0 mg/kg), DBP (5,000 ppm), and two or three combinations of these toxicants 6 h per day for 32- and 52-wk showed increases in the frequencies of TG rlymphocytes compared to the control groups. Additive interactions were noted from two combination groups compared to the ozone alone in both sexes of 32- and 52-wk studies. The most common specific mutation type in the hprt genes of test materials-treated male and female mice was transversion with very few transition. The results indicate that such dominant transversion may be responsible for toxicity and combined exposure to ozone, NNK, and DBP induces additive genotoxicities compared to ozone alone.

Comparison of Protein Expression in Normal Myometrium and Uterine Leiomyoma Using Two-Dimensional Gel Electrophoresis in Korean Women / 대한산부인과학회잡지

OBJECTIVE: Comparison of protein expression by two-dimensional gel electrophoresis (2-DE) in normal myometrium and uterine leiomyoma in Korean women. METHODS: Normal myometrium and uterine leiomyoma tissues were solubilized with 2-DE buffer and the first dimension of PROTEAN IEF CELL, isoelectric focusing (IEF), was performed using pH4-8 linear IPG strips of 17 cm. And then running 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) and sliver stain. Scanned image analyzed using PDQuest 2-D softwareTM. Protein spot spectrum was identified by assisted laser desorption/ionization-time of fighting (MALDI-TOF) and the protein mass spectrums identification were performed by searching protein databases of Swiss-prot/TrEMBL, Mascot and MS-FIT. RESULTS: In this study, we found 17 up-regulated proteins (phosphate carrier protein, 60 kDa heat shock protein, acidic calcium-independent, glutathione transferase omega, chloride intracellular channel 4, Ras-related protein Rab-11B, phosphatidylinositol transfer protein alpha isoform, type II keratin subunit protein, Cofilin 2 isoform 1, transgelin, ATP carrier protein, alpha-catenin homolog, parkinson disease 2, apo-cellular retinoic acid binding protein II, osteoglycin preproprotein, proteasome activator subunit 1 isoform, Unnamed protein) and 7 down-regulated proteins (Serum amyloid P component, annexin IV, alpha 1 actin precursor, hypoxanthine-guanine phosphoribosyltransferase, tumor necrosis factor receptor superfamily member EDAR precursor, peroxiredoxin 2, translation elongation factor EF-Tu precursor) between myometrium and leiomyoma. CONCLUSION: 2-DE offer total protein expression between normal myometrium and uterine leiomyoma, and searching of differently expressed protein for the diagnostic markers of leiomyoma.

A Case of Lesch-Nyhan Syndrome

Lesch-Nyhan syndrome is an X-linked recessive disorder characterized by hyperuricemia, choreoathetosis, spasticity, mental retardation, and compulsive, self-injurious behavior. This disorder results from a complete deficiency of the purine salvage enzyme, hypoxanthine-guanine phosphoribosyl transferase(HPRT). We report here on a case of Lesch-Nyhan syndrome in a 1-year, 7-month-old male who presented with frequent vomiting, failure to thrive, and developmental delay. The diagnostic work-up revealed hyperuricemia, hyperuricosuria, and medullary nephrolithiasis. The HPRT activity in the erythrocytes was undetectable with a biochemical assay. We also identified de novo mutation which was a deletion of the 649th base, adenosine, in HPRT gene(649delA) by analysis of cDNA using RT-PCR technique coupled with direct sequencing.

Partial HPRT Deficiency Due to a Missense Mutation in the HPRT Gene

An 8-month-old male infant presented with persistent, gross, orange-colored crystals in his urine. His physical and neurological development was normal. Laboratory study showed hyperuricemia, hyperuricosuria and urate crystaluria. He was determined to have partial hypoxanthine-guanine phosphoribosyl transferase(HPRT) deficiency. The molecular genetic analysis revealed a missense mutation in the patient's HPRT gene. By sequencing the patient's cDNA, we identified an A-to-G transition at nucleotide 239, resulting in the replacement of Aspartate with Glycine at amino acid 80 in the HPRT. To our knowledge, this mutation has not previously been reported. Our patient is now being placed on allopurinol therapy, and has had no problem since. Partial HPRT deficiency has been known to cause recurrent acute renal failure without the phenotypic features of Lesch-Nyhan syndrome. Therefore, we think that early diagnosis and treatment are very crucial in preventing acute renal failure.

Labeling embryonic stem cells with enhanced green fluorescent protein on the hypoxanthineguanine phosphoribosyl transferase locus / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To label embryonic stem (ES) cells with enhanced green fluorescent protein (EGFP) on the hypoxanthineguanine phosphoribosyl transferase (HPRT) gene locus for the first time to provide a convenient and efficient way for cell tracking and manipulation in the studies of transplantation and stem cell therapy.</p><p><b>METHODS</b>Homologous fragments were obtained by polymerase chain reaction (PCR), from which the gene targeting vector pHPRT-EGFP was constructed. The linearized vector was introduced into ES cells by electroporation. The G418(r)6TG(r) cell clones were obtained after selection with G418 and 6TG media. The integration patterns of these resistant cell clones were identified with Southern blotting.</p><p><b>RESULTS</b>EGFP expressing ES cells on the locus of HPRT were successfully generated. They have normal properties, such as karyotype, viability and differentiation ability. The green fluorescence of EGFP expressing cells was maintained in propagation of the ES cells for more than 30 passages and in differentiated cells. Cultured in suspension, the "green" ES cells aggregated and formed embryoid bodies, retaining the green fluorescence at varying developmental stages. The "green" embryoid bodies could expand and differentiate into various types of cells, exhibiting ubiquitous green fluorescence.</p><p><b>CONCLUSIONS</b>This generation of "green" targeted ES cells is described in an efficient protocol for obtaining the homologous fragments by PCR. Introducing the marker gene in the genome of ES cells, we should be able to manipulate them in vitro and use them as vehicles in cell-replacement therapy as well as for other biomedical and research purposes.</p>

Genotoxic and nongenotoxic effects of glycidyl methacrylate on human lung fibroblast cells / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To evaluate the genotoxic and nongenotoxic effects of short-term exposure to glycidyl mathacrylate (GMA) on human lung fibroblast cells (2BS cells) in vitro.</p><p><b>METHODS</b>DNA strand breakage was determined by single cell gel electrophoresis, and DNA ladder formation assay and flow cytometric analysis were carried out to detect apoptic responses of cells to GMA exposure. The HPRT gene mutation assay was used to evaluate the mutagenicity, and the effect of GMA on gap junctional intercellular communication (GJIC) in the exposed cells was examined with the scrape loading/dye transfer technique. The ability of GMA to transform 2BS cells was also tested by an in vitro cell transformation assay.</p><p><b>RESULTS</b>Exposure to GMA resulted in a dose-dependent increase in DNA strand breaks but not apoptic responses. GMA was also shown to significantly induce HPRT gene mutations and morphological transformation in 2BS cells in vitro. In contrast, GMA produced a concentration-dependent inhibition of GJIC.</p><p><b>CONCLUSIONS</b>GMA elicits both genotoxic and nongenotoxic effects on 2BS cells in vitro. The induction of DNA damage and gene mutations and inhibition of GJIC by GMA may casually contribute to GMA-induced cell transformation.</p>

Comparison of Hypoxanthine-Guanine Phosphoribosyltransferase Activity between Normal and Gout Patients / 대한류마티스학회지

OBJECTIVE: It has been known that the enzyme deficiency associated with uric acid production is the minor cause of gout. The purpose of this study is to evaluate the role of hypoxanthine- guanine phosphoribosyltransferase (HGPRT) activity deficiency in the development of gout. METHODS: Assay of HGPRT activity was performed on 38 gout patients and 107 controls compromising 82 men and 25 women. In the patients with gout, age of onset, sex, family history of gout, duration of disease, serum uric acid level, and uric acid concentration of 24-hour urine were analyzed. RESULTS: In gout patients and control male group, the HGPRT activity was lowest in the age of forties. But, in female, there was no decreasing tendency with aging. In 4 gout patients, HGPRT activities were severely deficient and their ages of onset were less than 30 years old, and all have a family history of gout. CONCLUSION: In men, the lowest HGPRT activity in the age of forties could be one of the factors that make the onset of gout is highest in the fifth decade of age. The deficiency of HGPRT enzyme and family history of gout are the important factors in the pathogenesis of early onset gout.

Study on the silica-induced cytotoxicity and hprt gene mutagenisis in rat lung fibroblasts and alveolar type II epithelial cells / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To explore the differences of the silica-induced inhibition on cellular proliferation and hprt gene mutagenesis between lung fibroblasts and alveolar type II cells.</p><p><b>METHODS</b>The proliferation inhibitive cytotoxicity was detected by MTT (3-[4,5-Dimethylthiazolzyl]-2,5-Diphenyl Tetrazolium Bromide) colorimetric method. Mutation in the hprt gene was screened by culture in the presence of the toxic purine analog, 6-thioguanine (6-TG).</p><p><b>RESULTS</b>Under the same circumstances of silica exposure, alveolar type II cells was more sensitive than lung fibroblasts for proliferation inhibition. The median proliferation inhibition concentration (IC50) of silica on epithelial was 140 micrograms/cm2, whereas IC50 of silica on fibroblasts was 282 micrograms/cm2. At the same doses of silica, the hprt gene mutation frequency in type II cells (84.2 x 10(-6))-156.6 x 10(-6) was statistically higher than that in fibroblasts (67.6 x 10(-6)-114.3 x 10(-6), P < 0.05).</p><p><b>CONCLUSION</b>There were significant differences of both silica-induced cell proliferation inhibition and hprt gene mutation between rat lung fibroblasts and type II epithelial cells. In vitro, cultured rat alveolar type II cells were more sensitive in cytotoxicity and hprt gene mutagenesis to silica dust than lung fibroblasts were.</p>

Effect of ethylnitrosourea on HPRT gene in human promyelocytic leukemia cells / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the molecular spectra and mechanism of human hypoxanthine guanine phosphoribosyl transferase (HPRT) gene mutation induced by ethyluitrosourea (ENU).</p><p><b>METHODS</b>Single cell cloning culture, two-way screening, multiple PCR amplification and electrophoresis technique were used.</p><p><b>RESULTS</b>With dose of ENU increasing, cell plating efficiency reduced (in the group with 100-200 micro g/ml doses, P < 0.01) and mutation frequency increased (in the group with 12.5-200.0 micro g/ml doses, P < 0.05) significantly. There was no all exons deletion in spontaneous mutations, and only 7.7% of them were detected as single exon deletion. But, deletion was found in 79.7% of ENU-induced mutations (62.5%-89.4%, P < 0.01), and deletion mutations in all nine exons of HPRT gene. Most of ENU-induced mutations were chain deletion with multiple exons (88.1%).</p><p><b>CONCLUSIONS</b>The spectra in spontaneous mutations differed completely from ENU-induced ones. ENU was liable to cause bigger changes in genetic structure, which suggested a stronger ENU's mutagenesis.</p>

Uma nova estratégia para o diagnóstico de mulheres portadoras de mutações que causam a síndrome de LESCH-NYHAN

A síndrome de Lesch-Nyhan é uma entidade clínica que apresenta um padrão de herança recessivo ligado ao cromossomo X. Pertence ao grupo dos erros inatos do metabolismo e é originada por uma deficiência absoluta da enzima hipoxantina-guanina fosforibosiltransferase (HPRT1; EC 2.4.2.8). Mulheres heterozigotas para mutações HPRT1 que causam a síndrome de Lesch-Nyhan podem ser detectadas mediante análises moleculares de híbridos celulares somáticos derivados da fusão de Iinfócitos de sangue periférico e células Hprt-negativas provenientes de roedor com a subseqüente seleção em meio de cultura contendo hipoxantina, aminopterina e timidina (HAT). A origem parental do cromossomo X contendo o alelo HPRT1 normal em linhagens celulares híbridas HPRT1 + pode ser determinada por haplótipos moleculares demonstrados por marcadores altamente polimórficos ligados ao cromossomo X. Esta metodologia foi utilizada para estudar uma possível heterozigota cujo cromossomo X paterno ativo esta sempre presente nas linhagens celulares derivadas dela. Contrariamente, o cromossomo X materno esteve sistematicamente ausente na maioria dos híbridos, e quando presente, estava inativo coexistindo com um X paterno ativo. Sub-clones sem o X paterno ativo e contendo o X materno inativo, não apresentaram atividade HPRT1 quando tratados com 5-aza-citidina. A ausência de transcrito do HPRT1 após o tratamento foi comprovada mediante RT-PCR. Estes resultados claramente demonstraram que a probanda é portadora de uma mutação responsável pela deficiência de HPRT1

Lesch-Nyhan syndrome is a recessive X-Iinked metabolic disorder resulting from absolute deficiency of the enzyme hypoxanthine/guanine phosphoribosy/transferase (HPRT1; EC 2.4.2.8). Heterozygous carriers of HPRT1 mutations responsible for Lesch-Nyhan syndrome can be detected by analysis of somatic cell hybrids derived from peripheral blood Iymphocytes and Hprt-negative cells of rodent origin followed by selection in culture medium containing hypoxanthine, aminopterine, and thymidine (HAT). The parental origin of the X chromosome containing the normal HPRT1 allele in HPRT1 + hybrid cell lines can be determined by molecular haplotyping attributable to highly polymorphic X-Iinked markers. We used this procedure to study a presumed carrier whose paternal active X chromosome always segregated in the cell hybrids derived from her. Conversely, her maternal X chromosome was systematically absent in most of cell hybrids, or when present, it was inactive and coexisted with an active, paternal X chromosome. Sub-clones lacking the paternal, active X chromosome and containing the X inactive maternal chromosome were unable to reactivate the HPRT1 locus following treatment with 5-aza-eytidine. HPRT1 transcripts were found to be absent with RT -PCR. These results c1early demonstrated that the proband was a heterozygous carrier of a mutation responsible for HPRT1 deficiency