The effects of acute responsive high frequency stimulation (HFS) to the subiculum on seizures and interictal spikes were investigated in a semi-acute kainic acid (KA) induced seizure model in rats. Wistar rats (n = 15) were implanted with an electrode-cannula complex in the CA3 area, stimulation and recording electrodes in the subiculum and another recording electrode at the contralateral motor cortex. Two weeks later rats were injected repeatedly with KA (0.05 μg/0.1 μL) for 3 days with an interval of 48 h. HFS (125 Hz, 100 μsec) was delivered to the subiculum at a predetermined intensity range (100–500 μA) in the HFS group (n = 7) when seizures were visually detected, while no stimulation was delivered in the sham control group (n = 8). Various severities of seizures were obtained (Stage I–V) and all rats of both groups reached Stage V (Racine's scale) on Day 1. The HFS group had less focal seizures and a longer inter-focal seizure interval on Day 1. Interictal spike rate was also lower in the HFS group and decreased with injection days. Significant day effects were found for the latency, number of focal seizures, and duration of focal seizures and generalized seizures while differences between groups were no longer present. Responsive HFS did not disrupt ongoing seizures. However, focal seizures and interictal spikes were suppressed by HFS. Such anticonvulsant effects of acute subicular stimulation indicate that the subiculum is involved in seizure generation. The reduction of seizure sensitivity over the injection day reflects an intrinsic anticonvulsant mechanism.

Tumor hypoxia is correlated with genetic alteration and malignant progression. Our previous studies indicated that the hypoxia-inducible transcription factor, HIF-1α, is responsible for hypoxic suppression of DNA repair in tumor cells by a non-canonical mode of action that requires the HIF-1α PAS-B subdomain. The involvement of HIF-1α in genetic alteration has raised an intriguing question as to whether normal cells would respond to hypoxic stress differently to avert genetic alteration. In this study, we chose several mouse cell types ranging from benign to malignant, apoptosis-proficient to apoptosis-deficient, and determined their responses to HIF-1α expression. In agreement with our previous findings, transient hypoxia and HIF-1α expression inhibited DNA repair and induced DNA damage in all cell types examined; however, cumulative DNA damage only occurred in apoptosis-deficient, malignant cells transduced for sustained expression of HIF-1α or HIF-1α PAS-B itself. In keeping with the theory of apoptosis as a cancer barrier, only these apoptosis-deficient cells acquired anchorage-independent growth and epithelial-mesenchymal transition. Furthermore, these cells exhibited increased Akt activity and resistance to etoposide by inhibiting autophagy. Altogether, our results define an essential role for apoptosis to prevent HIF-1α-induced genetic alteration and thereby malignant progression.

Brown coat colour has been described in Chinese-Tibetan, Kele, and Dahe pigs. Here, we report the identification of a causal mutation underlying the brown colouration. We performed a genome-wide association study (GWAS) on Tibetan and Kele pigs, and found that brown colours in Chinese breeds are controlled by a single locus on pig chromosome 1. By using a haplotype-sharing analysis, we refined the critical region to a 1.5-Mb interval that encompasses only one pigmentation gene: tyrosinase-related protein 1 (TYRP1). Mutation screens of sequence variants in the coding region of TYRP1 revealed a strong candidate causative mutation (c.1484_1489del). The protein-altering deletion showed complete association with the brown colouration across Chinese-Tibetan, Kele, and Dahe breeds by occurring exclusively in brown pigs (n=121) and lacking in all non-brown-coated pigs (n=745) from 27 different breeds. The findings provide the compelling evidence that brown colours in Chinese indigenous pigs are caused by the same ancestral mutation in TYRP1. To our knowledge, this study gives the first description of GWAS identifying causal mutation for a monogenic trait in the domestic pig.

Hypoxia is known to favor tumor survival and progression. Numerous studies have shown that hypoxia-inducible factor 1α (HIF-1α), an oxygen-sensitive transcription factor, is overexpressed in various types of human cancers and upregulates a battery of hypoxia-responsive genes for the growth and survival of cancer cells. Although tumor progression involves the acquisition of genetic and/or epigenetic changes that confer additional malignant traits, the underlying mechanisms of these changes remain obscure. We recently identified an alternative mechanism of HIF-1α function by which HIF-1α suppresses DNA repair by counteracting c-Myc transcriptional activity that maintains gene expression. Here we demonstrate that this HIF-α–c-Myc pathway plays an essential role in mediating hypoxic effects on malignant progression via genetic alterations, resulting in formation of malignant tumors with aggressive local invasion and epithelial–mesenchymal transition. We show an absolute requirement of the HIF-α–c-Myc pathway for malignant progression, whereas the canonical transcription function of HIF-1α alone is insufficient and seemingly dispensable. This study indicates that HIF-1α induction of genetic alteration is the underlying cause of tumor progression especially by the hypoxic microenvironment.

Domestication, modern breeding and artificial selection have shaped dramatically the genomic variability of domestic animals. In livestock, the so-called FAT1 quantitative trait locus (QTL) in porcine chromosome 4 was the first QTL uncovered although, to date, its precise molecular nature has remained elusive. Here, we characterize the nucleotide variability of 13 fragments of ∼500 bp equally spaced in a 2 Mb region in the vicinity of the FAT1 region in a wide-diversity panel of 32 pigs. Asian and European animals, including local Mediterranean and international pig breeds, were sequenced. Patterns of genetic variability were very complex and varied largely across loci and populations; they did not reveal overall a clear signal of a selective sweep in any breed, although FABP4 fragment showed a significantly higher diversity. We used an approximate Bayesian computation approach to infer the evolutionary history of this SSC4 region. Notably, we found that European pig populations have a much lower effective size than their Asian counterparts: in the order of hundreds vs hundreds of thousands. We show also an important part of extant European variability is actually due to introgression of Asian germplasm into Europe. This study shows how a potential loss in diversity caused by bottlenecks and possible selective sweeps associated with domestication and artificial selection can be counterbalanced by migration, making it much more difficult the identification of selection footprints based on naive demographic assumptions. Given the small fragment analyzed here, it remains to be studied how these conclusions apply to the rest of the genome.

Background

This study describes a repeated measures prediction index to identify patients at high risk of ≥ grade 2 hand-foot skin reaction (HFSR) before each week of sorafenib therapy.

Methods

Data from 451 patients who received a sorafenib (400 mg bid) as part of a clinical trial were reviewed (Escudier B, Eisen T, Stadler WM et al. Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med 2007; 356: 125–134). Generalized estimating equations were used to develop the final risk model. A risk-scoring algorithm (range 0–58) was then derived from the final model coefficients. External validation was then carried out on a new sample of 1145 patients who received sorafenib under an expanded access program.

Results

Pretreatment white blood cell count, female gender, good performance status, presence of lung and liver metastases and number of affected organs were predictors for ≥ grade 2 HFSR. A nonlinear association between HFSR risk and treatment duration was also identified where risk was maximized at week 5 followed by a gradual decline. Before each week of therapy, patients with risk scores >40 would be considered at high risk for developing ≥ grade 2 HFSR.

Conclusions

The application and planned continued refinement of this prediction tool will be an important source of patient-specific risk information for the development of moderate to severe HFSR.

Summary

The physiological regulation of the red cell mass depends upon enhanced transcription of the erythropoietin (Epo) gene in response to hypoxia. Studies of Epo gene expression have been useful in investigating the mechanism by which cells and tissues sense hypoxia and respond with biologically appropriate alterations in gene expression. It is likely that oxygen sensing involves a heme protein in which cobalt and nickel can substitute for iron in the porphyrin ring. Indirect evidence suggests that the sensor is present in all cells and is a multi-subunit assembly containing an NAD(P)H oxidase capable of generating peroxide and reactive oxygen intermediates, which serve as signaling molecules. The up-regulation of Epo gene transcription by hypoxia is mediated by at least two known DNA-binding transcription factors, hypoxia-inducible factor 1 (HIF-1) and hepatic nuclear factor 4 (HNF-4), which bind to cognate response elements in a critical 3′ enhancer approximately 50 bp in length. HIF-1 binding is induced by hypoxia as well as by cobalt. The activation of HIF-1 by hypoxia depends upon the selective protection of its α subunit from ubiquitin-dependent proteolysis by means of a mechanism that involves redox chemistry and perhaps phosphorylation. HNF-4 is an orphan nuclear receptor that is constitutively expressed in kidney and liver and which cooperates with HIF-1 to give maximal hypoxic induction. In hypoxic cells, p300 or a related family member forms a macromolecular assembly with HIF-1 and HNF-4, enabling transduction from the Epo 3′ enhancer to the apparatus on the promoter responsible for the initiation of transcription.

Bleach digestion of sputum prior to smear preparation has been reported to increase the yield of microscopy for diagnosing pulmonary tuberculosis, even in high-HIV-prevalence settings. To determine the diagnostic accuracy of bleach microscopy, we updated a systematic review published in 2006 and applied the Grading of Recommendations Assessment, Development, and Evaluation framework to rate the overall quality of the evidence. We searched multiple databases (as of January 2009) for primary studies in all languages comparing bleach and direct microscopy. We assessed study quality using a validated tool and heterogeneity by standard methods. We used hierarchical summary receiver operating characteristic (HSROC) analysis to calculate summary estimates of diagnostic accuracy and random-effects meta-analysis to pool sensitivity and specificity differences. Of 14 studies (11 papers) included, 9 evaluated bleach centrifugation and 5 evaluated bleach sedimentation. Overall, examination of bleach-processed versus direct smears led to small increases in sensitivity (for bleach centrifugation, 6% [95% confidence interval {CI} = 3 to 10%, P = 0.001]; for bleach sedimentation, 9% [95% CI = 4 to 14%, P = 0.001]) and small decreases in specificity (for bleach centrifugation, −3% [95% CI = −4% to −1%, P = 0.004]; for bleach sedimentation, −2% [95% CI = −5% to 0%, P = 0.05]). Similarly, analysis of HSROC curves suggested little or no improvement in diagnostic accuracy. The quality of evidence was rated very low for both bleach centrifugation and bleach sedimentation. This updated systematic review suggests that the benefits of bleach processing are less than those described previously. Further research should focus on alternative approaches to optimizing smear microscopy, such as light-emitting diode fluorescence microscopy and same-day sputum collection strategies.

We have taken the opportunity of a clinical trial of the potential efficacy and safety of FK 506 (tacrolimus) in chronic progressive multiple sclerosis (MS) to examine the influence of this potent new immunosuppressant on circulating T-lymphocytes in an otherwise healthy non-transplant population. Peripheral blood levels of subsets of CD4+ T lymphocytes expressing the activation molecule interleukin-2 receptor (p55 &α chain: CD25) or the CD45RA isoform were determined sequentially in 19 patients that were treated continuously with oral FK 506 (starting dose 0.15 mg/kg/day) for 12 months. No significant change in the proportion of circulating CD25+CD4+ cells was observed over the study period in which the mean trough plasma FK 506 level rose from 0.3 ± 0.2 to 0.5 ± 0.4 ng/ml. There was also no significant effect of FK 506 on the percentage of CD45RA+CD4+ cells in the peripheral blood at 12 months compared with pretreatment values. Analysis of a subgroup of 7 patients, who showed a sustained reduction in CD25+ CD4+ cells and a reciprocal increase in CD45RA+ CD4+ cells for at least 6 months after start of treatment, did not reveal any difference in disability at one year compared with the treatment group as a whole. The side effects of FK 506 were mild and the overall degree of disability estimated by the mean Kurtzke expanded disability status scale (EDSS) score or the ambulation index did not deteriorate significantly in the 19 patients studied over the 12 months of FK 506 administration.

Angiogenesis has long been recognized as an essential element in tumor growth. Since the conception of antiangiogenesis for cancer therapeutics, great strides have been made in understanding the molecular biology underlying angiogenesis, both in cancer and in physiology. By capitalizing on these advancements through bench-to-bedside research, potent antiangiogenic agents have been developed and tested. To date, the clinical results of most of these antiangiogenic agents have not met expectations. Even with the most successful agents, such as bevacizumab, used either as single agents or in combination with chemotherapy, gains in overall survival of cancer patients have been modest in most cases. In this article, the authors present the evolving views of antiangiogenic therapy, review recent experimental and clinical studies on antiangiogenesis, and address the fundamental role of hypoxia in tumor progression, which may be key to improving the efficacy of antiangiogenic therapy.

We have identified a novel function for a member of the transient receptor potential (TRP) protein super-family, TRPM2, in prostate cancer cell proliferation. TRPM2 encodes a non-selective cation-permeable ion channel. We found that selectively knocking down TRPM2 with the small interfering RNA technique inhibited the growth of prostate cancer cells but not of non-cancerous cells. The subcellular localization of this protein is also remarkably different between cancerous and non-cancerous cells. In BPH-1 (benign), TRPM2 protein is homogenously located near the plasma membrane and in the cytoplasm, whereas in the cancerous cells (PC-3 and DU-145), a significant amount of the TRPM2 protein is located in the nuclei in a clustered pattern. Furthermore, we have found that TRPM2 inhibited nuclear ADP-ribosylation in prostate cancer cells. However, TRPM2 knockdown-induced inhibition of proliferation is independent of the activity of poly(ADP-ribose) polymerases. We conclude that TRPM2 is essential for prostate cancer cell proliferation and may be a potential target for the selective treatment of prostate cancer.

Background:

Pazopanib has shown clinical activity against multiple tumour types and is generally well tolerated. However, isolated elevations in transaminases and bilirubin have been observed. This study examined polymorphisms in molecules involved in pharmacokinetic and pharmacodynamic pathways of pazopanib and their association with hepatic dysfunction.

Methods:

Twenty-eight polymorphisms in 11 genes were evaluated in pazopanib-treated renal cell carcinoma patients. An exploratory analysis was conducted in 116 patients from a phase II study; a replication study was conducted in 130 patients from a phase III study.

Results:

No polymorphisms were associated with alanine aminotransferase elevation. The Gilbert's uridine-diphosphoglucuronate glucuronosyltransferase 1A1 (UGT1A1) TA-repeat polymorphism was significantly associated with pazopanib-induced hyperbilirubinemia in the phase II study. This association was replicated in the phase III study (P<0.01). Patients with TA6/TA6, TA6/TA7, and TA7/TA7 genotypes experienced median bilirubin increases of 0.31, 0.37, and 0.71 × upper limit of the normal range (ULN), respectively. Of the 38 patients with hyperbilirubinemia (⩾1.5 × ULN), 32 (84%) were either TA7 homozygotes (n=18) or TA7 heterozygotes (n=14). For TA7 homozygotes, the odds ratio (95% CI) for developing hyperbilirubinemia was 13.1 (5.3–32.2) compared with other genotypes.

Conclusions:

The UGT1A1 polymorphism is frequently associated with pazopanib-induced hyperbilirubinemia. These data suggest that some instances of isolated hyperbilirubinemia in pazopanib-treated patients are benign manifestations of Gilbert's syndrome, thus supporting continuation of pazopanib monotherapy in this setting.

Background

Surgical closure of patent ductus arteriosus (PDA) with severe pulmonary arterial hypertension in adults carries higher risk than in children.

Objectives

To investigate the application of self‐expandable occluders for transcatheter closure of PDA associated with severe pulmonary arterial hypertension in adults, and the assessment of immediate and short‐term results.

Methods

29 adult patients (6 men, 23 women) underwent attempted transcatheter closure of PDA at a mean (standard deviation (SD)) age of 31.1 (11.4) years (range 18–58 years) and a mean (SD) weight of 54.1 (7.1) kg (range 42–71 kg). On the basis of haemodynamic and clinical data obtained before and after trial occlusion, the final duct occlusion was determined and carried out. Radiographs of the chest, electrocardiograms and echocardiograms were used for follow‐up evaluation of the treatment within 1 day, 1 month and 3–6 months after successful closure.

Results

20 of the 29 patients had successful occlusion (group 1), and 9 patients failed (named group 2). In group 1, in which occlusion was successful, mean (SD) pulmonary arterial pressures decreased markedly after trial occlusion: 78 (19.3) mm Hg (range 50–125 mm Hg) before occlusion and 41 (13.8) mm Hg (range 23–77 mm Hg) after occlusion. Systemic arterial oxygen saturation was found to be >90% in 19 patients and <90% in the remaining patient before inhalation of oxygen, and >95% during inhalation of oxygen or after occlusion in all 20 patients. In group 2, the occlusion was not successful, because in two patients the device was not available; another two patients showed worsening of symptoms. The other five patients showed increased pulmonary arterial pressures after trial closure; their mean (SD) pulmonary arterial pressures increased by 10.3 (6) mm Hg (4–16 mm Hg) after trial occlusion, and systemic arterial oxygen saturation was 85.5% (2.6%) (range 82.6–88%) before inhalation of oxygen and 94.7% (1.7%) (range 90.7–99.1%) during inhalation of oxygen. In group 1, the dimensions of the left atrium, left ventricle and pulmonary artery increased considerably in 3–6‐months of follow‐up compared with those of preocclusion.

Conclusions

Transcatheter closure is an effective treatment for adults with PDA associated with reversible severe pulmonary arterial hypertension. Further research is needed for the evaluation of long‐term results.

Ryanodine receptors (RyRs) are one of the intracellular calcium channels involved in regulation of intracellular free calcium concentration ([Ca2+]i). The immunolocalization of RyRs was investigated in the developing rat cochlea at different postnatal days (PND). The change of [Ca2+]i in isolated outer hair cells (OHCs) was determined. Morphological results showed low expression of RyRs in the Kolliker’s organ from the PND 5 group. RyR expression in inner hair cells (IHCs) increased as the rats aged, and was mature after PND 14. RyRs in OHCs were expressed near the synaptic area of afferent and efferent nerves. RyRs in supporting cells were expressed widely and strongly. The application of ACh, ryanodine + ACh, and thapsigargin + ACh could induce a significant increase in [Ca2+]i in OHCs in the presence of extracellular calcium. This increase of [Ca2+]i induced by ACh was caused by not only the calcium influx through surface calcium channels, but also the calciuminduced calcium release (CICR) from intracellular RyR-sensitive calcium stores. Morphological and Ca imaging results suggested that RyRs expression is related to cochlear maturity, and may play an important role in its function.

Objectives:

Exposure to second-hand smoke (SHS) is widespread in restaurants in Ulaanbaatar, the capital city of Mongolia. While a smoke-free policy is the most effective way of protecting restaurant workers and customers from SHS, this has not been well accepted in Mongolia. Furthermore, little is known about restaurants’ attitude toward the smoke-free policy.

Methods:

A cross-sectional survey directed to restaurant owners or managers was conducted in 475 representative restaurants in Ulaanbaatar. Face-to-face interviews using a questionnaire and on-site observation were performed.

Results:

Only 29.3% of the restaurants claimed to prohibit smoking; none of the remaining had any protection toward SHS, and half of the restaurants estimated that more than 20% of customers would smoke inside. None of them had visible “no smoking” signs and the majority never received complaints about SHS. Despite the generally high level of knowledge of the health effects of SHS, of the 336 restaurants that were not smoke free, only 25.9% expressed that they planned to take action in the near future. By contrast, 87.8% of restaurants would support the government if it asked all restaurants to ban smoking. Multivariate analysis identified that restaurants having menus in foreign languages, selling cigarettes and predicting business decline were less likely to support the government smoke-free policy.

Conclusions:

This survey demonstrates that restaurants owners and managers were reluctant to take action on their own, but would support government policy. The government can assume a stronger role first by revising the law on tobacco control following the Framework Convention on Tobacco Control guideline.

Objective

To study the three dimensional (3D) reconstruction and 3D visualisation of the pancreas and create anatomy of the digitalised visual pancreas so as to construct a concrete basis for virtual operation and surgical operation on pancreas.

Methods

The digital imaging data of pancreas, duodenum, common bile duct, arteries, and veins were obtained from the virtual Chinese human—female 1 (VCH‐F1). The image data were investigated and 380 images ascertained of pancreas picked up from images numbers 2617 to 2996. Finally, the images from number 2574 to 3017 were adopted to segment and processed using ACDSee and Photoshop so as to reconstruct 3D pancreas digitally. The data of pancreatic surfaces were transformed into Visualization Toolkit (VTK). The GUI program written with VC+ was used to display this VTK file and realise 3D visualisation of the pancreas.

Results

3D reconstruction and visualisation of the pancreas and the peri‐pancreatic structures (the duodenum, the common bile duct,the inferior vena cava, the portal vein vessels, the aorta, the coeliac trunk vessels) was successful. The 3D and visualised pancreas manifested itself with its complete structure as well as its adjacency to other tissues.

Conclusion

The 3D reconstruction and 3D visualisation of the pancreas based on the digital data of VCH‐F1 produces a digitally visualised pancreas, which promises a novel method for virtual operation on the pancreas, clinical operation on the pancreas, and anatomy of 3D visualised pancreas.

Nicotine, the major psychoactive ingredient in tobacco interacting with nicotinic acetylcholine receptors (nAChR), is believed to have neuroprotective and neurotoxic effects on the developing brain. Neurotoxicity has been attributed to activation of homomeric α7 nAChRs, neuroprotection to heteromeric α4β2 nAChRs. Thus, developmental nicotine could have opposite effects in different brain regions, depending on nAChR subtype expression. Here, we determined if chronic neonatal nicotine exposure (CNN), during a period of brain growth corresponding to the third human trimester, differentially regulates nAChR expression, cell death, and morphological properties in hippocampus and cerebellum, two structures maturing postnatally. Rat pups were orally treated with 6 mg/kg/day nicotine from postnatal day (P)1 to P7. On P8, expression for α4, α7 and β2 mRNA was determined by in situ hybridization; nAChR binding sites in by receptor autoradiography, dying neurons by TUNEL and Fluoro-Jade staining and morphological properties by analysis of Cresyl-Violet stained sections. In control cerebellum, strong expression of α4, β2 mRNA and heteromeric nAChRs labeled with [125I]-Epibatidine were found in granule cells, and α7 mRNA and homomeric nAChRs labeled with [125I]-αBungarotoxin in the external germinal layer. In control hippocampus, low expression of α4 mRNA and heteromeric nAChRs and high expression of α7 mRNA and homomeric nAChRs were detected. CNN increased heteromeric nAChR binding in hippocampus but not cerebellum and significantly decreased neuronal soma size and increased packing density in hippocampal principal cells but not in cerebellum. CNN did not increase the number of dying cells in any area, but significantly fewer TUNEL-labeled cells were found in CA3 strata oriens and radiatum and cerebellar granule layer. Thus, the hippocampus seems to be more sensitive than the cerebellum to CNN which could result from different nAChR subtype expression and might explain long-lasting altered cognitive functions correlated with gestational nicotine exposure due to changes in hippocampal cell morphology.

Administration of donor-derived immature dendritic cells (DC) treated with transforming growth factor-beta (TGF-β) to prevent allograft rejection is not applicable for clinical use. We therefore attempted to explore the use of recipient-derived DC pulsed with donor antigens via the indirect pathway (cross-priming). DC were propagated from C3H (H2k) bone marrow (BM) with GM-CSF and IL-4. TGF-β (0.2ng/ml) was added at the initiation of culture and the resultant (TGF-β DC) were pulsed with B10 (H2b) splenocyte lysate. Expression of MHC class I and II were not affected, while CD40, CD80 and CD86 co-stimulatory molecules on DC was significantly inhibited by treatment with TGF-β. C3H DC pulsed with B10 antigens stimulated proliferate responses in C3H T cells was inhibited when DC were treated with TGF-β, and the CTL activity was also inhibited. This correlated with reduced IFN-γ and increased IL-10 production. A single injection of TGF-β DC prolonged allograft survival (MST 18 days vs. 10 days in no-DC treatment control, p<0.05). These data indicate that an approach utilizing recipient DC as a “vaccine” strategy is possible.

Objective: To characterize the frequency and severity of incidental findings in brain MRIs of young and older adult research volunteers, and to provide an evaluation of the ethical challenges posed by the detection of such findings. Methods: The authors reviewed 151 research MRI scans obtained retrospectively from subjects recruited to studies as healthy volunteers. Incidental findings were classified into four categories: no referral, routine, urgent, or immediate referral. p Values for significance were computed from χ2 tests of contingency. Results: Of 151 studies, the authors found an overall occurrence of incidental findings having required referral of 6.6%. By age, there were more findings in the older cohort (aged >60 years) than in the younger cohort (p < 0.05) and in more men than women in the older cohort (p < 0.001). Three of four (75%) findings in the younger cohort were classified in the urgent referral category; 100% of the findings in the older cohort were classified as routine (p < 0.05). Conclusion: The significant presence but different characteristics of incidental findings in young and older subjects presumed to be neurologically healthy suggest that standards of practice are needed to guide investigators in managing and communicating their discovery.

The results of recent studies suggest that the mouse Sac (saccharin preference) locus is identical to the Tas1r3 (taste receptor) gene. The goal of this study was to identify Tas1r3 sequence variants associated with saccharin preference in a large number of inbred mouse strains. Initially, we sequenced ~6.7 kb of the Tas1r3 gene and its flanking regions from six inbred mouse strains with high and low saccharin preference, including the strains in which the Sac alleles were described originally (C57BL/6J, Sacb; DBA/2J, Sacd). Of the 89 sequence variants detected among these six strains, eight polymorphic sites were significantly associated with preferences for 1.6 mm saccharin. Next, each of these eight variant sites were genotyped in 24 additional mouse strains. Analysis of the genotype–phenotype associations in all 30 strains showed the strongest association with saccharin preference at three sites: nucleotide (nt) −791 (3 bp insertion/deletion), nt +135 (Ser45Ser), and nt +179 (Ile60Thr). We measured Tas1r3 gene expression, transcript size, and T1R3 immunoreactivity in the taste tissue of two inbred mouse strains with different Tas1r3 haplotypes and saccharin preferences. The results of these experiments suggest that the polymorphisms associated with saccharin preference do not act by blocking gene expression, changing alternative splicing, or interfering with protein translation in taste tissue. The amino acid substitution (Ile60Thr) may influence the ability of the protein to form dimers or bind sweeteners. Here, we present data for future studies directed to experimentally confirm the function of these polymorphisms and highlight some of the difficulties of identifying specific DNA sequence variants that underlie quantitative trait loci.

The cellular response to hypoxia is, at least in part, mediated by the transcriptional regulation of hypoxia-responsive genes involved in balancing the intracellular ATP production and consumption. Recent evidence suggests that the transcription factor, HIF-1α, functions as a master regulator of oxygen homeostasis by controlling a broad range of cellular events in hypoxia. In normoxia, HIF-1α is targeted for destruction via prolyl hydroxylation, an oxygen-dependent modification that signals for recognition by the E3 ubiquitin ligase complex containing the von Hippel-Lindau tumor suppressor (VHL). Three HIF prolyl hydroxylases (EGLN1, EGLN2, and EGLN3) have been identified in mammals, among which, EGLN1 and EGLN3, are hypoxia-inducible at their mRNA levels in a HIF-1α-dependent manner. In this study, we demonstrate that apart from promoting HIF-1α proteolysis in normoxia, EGLN1 specifically represses HIF-1α transcriptional activity in hypoxia. Ectopic expression of EGLN1 inhibited HIF-1α transcriptional activity without altering its protein levels in a VHL-deficient cell line, indicating a discrete activity of EGLN1 in transcriptional repression. Conversely, silencing of EGLN1 expression augmented HIF-1α transcriptional activity and its target gene expression in hypoxia. Hence, we propose that the accumulated EGLN1 in hypoxia acts as a negative-feedback mechanism to modulate HIF-1α target gene expression. Our finding also provides new insight into the pharmacological manipulation of the HIF prolyl hydroxylase for ischemic diseases.