Kinds and locations of mutations arising spontaneously in the coding region of theHPRT gene of finite-life-span diploid human fibroblasts

Spontaneous thioguanine-resistant mutants were derived from populations of finite-life-span, diploid human fibroblasts by means of a fluctuation analysis. cDNA was prepared from mutantHPRT mRNA and amplified by the polymerase chain reaction, and the sequence of the product was analyzed. Exon deletions, which very likely arose from mutations in the intron splice site consensus sequences, were found in 10 of the 37 mutants examined (27% of the total). Among the 28 mutations in the coding sequence, base pair substitutions predominated (89%). With the exception of one base pair involved in a tandem mutation, all base pair substitutions resulted in alterations in the predicted amino acid sequence of the protein. In addition there were three frameshift mutations, consisting of the deletion of one or two base pairs. Although mutations occurred throughout the coding sequence, 50% (14/28) were found in the 5 portion of exon 3.     

Heterogeneity of toxicant response: sources of human variability.

While risk assessment models attempt to predict human risk to toxicant exposure, in many cases these models cannot account for the wide variety of human responses. This review addresses several primary sources of heterogeneity that may affect individual responses to drug or toxicant exposure. Consideration was given to genetic polymorphisms, age-related factors during development and senescence, gender differences associated with hormonal function, and preexisting diseases influenced by toxicant exposure. These selected examples demonstrate the need for additional steps in risk assessment that are needed to more accurately predict human responses to toxicants and drugs.     

Disruption of rat forebrain development by glucocorticoids: critical perinatal periods for effects on neural cell acquisition and on cell signaling cascades mediating noradrenergic and cholinergic neurotransmitter/neurotrophic responses.

Glucocorticoids are the consensus treatment for the prevention of respiratory distress in preterm infants, but there is evidence for increased incidence of neurodevelopmental disorders as a result of their administration. We administered dexamethasone (Dex) to developing rats at doses below or within the range of those used clinically, evaluating the effects on forebrain development with exposure in three different stages: gestational days 17-19, postnatal days 1-3, or postnatal days 7-9. At 24 h after the last dose, we evaluated biomarkers of neural cell acquisition and growth, synaptic development, neurotransmitter receptor expression, and synaptic signaling mediated by adenylyl cyclase (AC). Dex impaired the acquisition of neural cells, with a peak effect when given in the immediate postnatal period. In association with this defect, Dex also elicited biphasic effects on cholinergic presynaptic development, promoting synaptic maturation at a dose (0.05 mg/kg) well below those used therapeutically, whereas the effect was diminished or lost when doses were increased to 0.2 or 0.8 mg/kg. Dex given postnatally also disrupted the expression of adrenergic receptors known to participate in neurotrophic modeling of the developing brain and evoked massive induction of AC activity. As a consequence, disparate receptor inputs all produced cyclic AMP overproduction, a likely contributor to disrupted patterns of cell replication, differentiation, and apoptosis. Superimposed on the heterologous AC induction, Dex impaired specific receptor-mediated cholinergic and adrenergic signals. These results indicate that, during a critical developmental period, Dex administration leads to widespread interference with forebrain development, likely contributing to eventual, adverse neurobehavioral outcomes.     

AAV-mediated delivery of the caspase inhibitor XIAP protects against cisplatin ototoxicity.

HYPOTHESIS: Delivery of the gene encoding X-linked inhibitor of apoptosis (XIAP) using an adeno-associated viral (AAV) vector can protect against cisplatin-mediated ototoxicity. BACKGROUND: Cisplatin is a widely used chemotherapeutic agent with significant ototoxic side effects. One possible mechanism of toxicity is apoptotic death of many cochlear cell types. Acute treatment with inhibitors of caspases- enzymes critical for apoptosis- has been shown to prevent hearing loss in vivo, but is too short-acting for therapeutic use. Gene therapy provides a specific and chronic means of delivering potential therapeutic gents. Introducing an anti-apoptotic gene into the cochlea could provide long-term prophylaxis against the ototoxic effects of cisplatin. METHOD: Two groups of rats were treated with unilateral injection into the round window of AAV harboring a gene encoding either XIAP or green fluorescent protein (GFP). After at least two months of gene expression, auditory-brainstem-response (ABR) threshold shifts and outer-hair-cell (OHC) number were measured in these two groups of animals after 72-hour treatment with cisplatin. RESULTS: Consistent with previous reports, uninjected and AAV.GFP-injected ears displayed profound ABR threshold elevations and OHC loss after cisplatin treatment. Ears that had been injected with AAV encoding XIAP, however, were significantly protected from these effects: cisplatin-induced ABR-threshold shift and hair-cell loss were attenuated by as much as 78% and 45%, respectively, when compared with contralateral (untreated) ears. CONCLUSION: XIAP delivery to the cochlea can protect against the audiometric changes and hair-cell loss associated with cisplatin ototoxicity. The efficacy, specificity, and duration of the protective effects make this a potentially attractive therapeutic paradigm.     

Molecular mechanisms of resistance to therapies targeting the epidermal growth factor receptor.

Targeted therapies that inhibit the activity of tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR) have shown activity against solid malignancies when used as single agents or in combination with chemotherapy. Although anti-EGFR therapies are active in some patients, eventually disease in nearly all patients will become refractory to therapy. Therefore, a better understanding of the mechanisms of resistance to anti-EGFR therapies is critical to further improve the efficacy of this class of agents. Mechanisms that mediate resistance to anti-EGFR therapies include the presence of redundant tyrosine kinase receptors, increased angiogenesis, and the constitutive activation of downstream mediators. Two recent landmark publications have also shown that specific mutations in the kinase domain of EGFR in some lung carcinomas are associated with markedly improved response rates to an EGFR tyrosine kinase inhibitor. Mutations in the EGFR receptor seem to play a significant role in determining the sensitivity of tumor cells to EGFR inhibitor therapy by altering the conformation and activity of the receptor. As the field of molecular therapeutics continues to evolve, a comprehensive understanding of resistance mechanisms will ultimately lead to refinements in our regimens to provide better care for patients with cancer.     

Aurone-derived 1,2,3-triazoles as potential fluorescence molecules in vitro

Aurones are a class of well-studied natural compounds primarily responsible for the yellow pigment in flowering plants and have been shown to have fluorescent properties as well as beneficial biological effects. Traditionally, aurones can be easily synthesized through a Knoevenagel condensation of benzofuranones with arylaldehydes. Recently, Kafle et al. unexpectedly synthesized a new aurone derivative containing a 1,2,3-triazole within its backbone. Since, 1,2,3-triazole containing structures have been shown to be useful as fluorophores with large Stokes shifts, we hypothesized that these new aurone-derived triazole compounds (ATs) could be utilized as potential fluorophores. Here we describe a newly-synthesized fluorescent compound which has potential for use as a live-cell probe, having a large Stokes shift of 118.3 ± 1.01 nm in phosphate-buffered saline with the benefit of increased fluorescence in protic environments, which is uncommon in aurone-derived fluorophores.

Aurones are a class of naturally occurring compounds with fluorescent derivatives. Here we show a newly synthesized derivative of aurones containing a 1,2,3-triazole which is fluorescent in aqueous environments and has potential to be used as a probe in vitro.     

Clinical potential of tensionlengthening strategies during nerve repair

<正>A (very) brief history of tension in nerve repair:Successful nerve repair is achieved by conveying as many axons successfully to their targets as possible.Typically,this is best achieved through a direct end-to-end repair under minimal tension (Millesi,1986).However,this is not feasible in most cases of trauma,where a segment of tissue damage must be excised and overcome.This has most commonly been addressed with the use of nerve grafts to bridge the gap.Autologous nerve grafts are cons...