Interaction between the <Emphasis Type="Italic">UCP2</Emphasis>–866G/A,mtDNA 10398G/A and <Emphasis Type="Italic">PGC1α</Emphasis> p.Thr394Thr and p.Gly482Ser polymorphisms in type 2 diabetes susceptibility in North Indian population

In the recent past, we have observed a possible role of 10398A and 16189C mtDNA and PGC1α p.Thr394Thr (rs2970847) and p.Gly482Ser (rs8192673) variant genotypes providing susceptibility/protection against type 2 diabetes mellitus (T2DM) in two North Indian population groups. These initial observations encouraged us to look at the candidate genes in combination with –866G/A (rs659366) polymorphism in uncoupling protein 2 (UCP2) in a single study of a relatively large sample size, constituted of both the cohorts, to unravel an interesting outcome of an additive interaction in-between the studied genes. In a total of 1,686 individuals (762 cases and 924 controls) belonging to Indo-European linguistic group from North India, a comparison of risk genotype combinations of: UCP2–866GG, mtDNA 10398A and PGC1α p.Thr394Thr or p.Gly482Ser against the protective genotypes: UCP2–866XA, mtDNA 10398G and PGC1α p.Thr394Thr (nominal P value = 1.75 × 10⁻¹⁴, Odds ratio, OR = 5.29, 3.40–8.22 at 95% CI) or PGC1α p.Gly482Ser (nominal p value = 4.42 × 10⁻²⁴, OR = 8.59, 5.53–13.35 at 95% CI), showed a highly significant difference and increased ORs. In a complex disease, it is always encouraging to find an additive interaction of multiple small effects of the studied candidate gene variations. An erratum to this article is available at .     

PPARGC1A基因Thr394Thr/Gly482Ser多态性与2型糖尿病的关联研究

对344例2型糖尿病患者和307名正常人的PPARGC1A基因单核苷酸多态性rs2970847(Thr394Thr)和rs8192678(Gly482Ser)与2型糖尿病的关系进行了单标记和单体型关联分析以及Logistic回归分析。在单标记分析中,对照组与病例组Thr394Thr的基因型和等位基因频率有显著差异(基因型, P =0.006; 等位基因, P < 0.001); Logistic回归和单体型分析表明, Thr394Thr的AA基因型及Thr394(ACA)-Ser482单体型增加患2型糖尿病的风险。Gly482Ser的基因型和等位基因频率在对照组与病例组间无显著差异。PPARGC1A基因是湖北汉人的一个2型糖尿病易感基因。     

PGC-1α Thr394Thr and Gly482Ser variants are significantly associated with T2DM in two North Indian populations: a replicate case-control study

The recent observations that Peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1A) is responsible for the induction of reactive oxygen species (ROS) detoxifying agents and that ROS triggers insulin resistance, support the role that this gene could play in the onset of Type 2 diabetes mellitus (T2DM). Two PGC1A variants Thr394Thr (rs2970847) and Gly482Ser (rs8192673) were genotyped in 822 subjects (351 T2DM cases and 471 controls) from two North Indian populations, represented as Group 1 (Kashmir population) and Group 2 (Punjab and Jammu population). Both Groups 1 and 2 showed a significant association of Thr394Thr variant with T2DM after applying Bonferroni corrections (P = 0.001 and 0.012, respectively). Logistic regression analysis for Thr394Thr susceptible genotypes together (rs2970847 G/A and A/A) conferred a 1.89-(95%CI 1.25–2.85) fold higher risk for T2DM in Group 1 and 1.81-(95%CI 1.19–2.78) fold risk in Group 2. The susceptible, Ser482 (rs8192673 G/A and A/A) genotypes, gave a 2.04 (95%CI 1.47–3.03) fold higher risk for T2DM in Group 1. Mitochondrial genotype backgrounds observed in association with T2DM (Bhat et al. 2007), when studied in combination with PGC1A variants, showed an increased prevalence in controls with mt10398G and 16189T along with G/G genotype background at the two polymorphic loci of PGC1A. These observations suggest that the two genotype backgrounds together could provide protection against T2DM. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article is available at .     

Apolipoprotein A1 −75 G/A and +83 C/T polymorphisms: susceptibility and prognostic implications in breast cancer

Apolipoprotein A1 (ApoA1) is the major apoprotein constituent of high-density lipoprotein that can play important roles in tumor invasion and metastasis. In the current report, we evaluated the role of the functional ApoA1 polymorphisms (−75 G/A and +83 C/T) as genetic markers for breast cancer susceptibility and prognosis. We used the polymerase chain reaction and restriction enzyme digestion (RFLP-PCR) to characterize the variations of the ApoA1 gene in 295 unrelated Tunisian patients with breast carcinoma and 197 healthy control subjects. No association was found between the +83 C/T genetic variation in ApoA1 gene and the risk of breast cancer occurrence. The presence of the (+83) T allele appeared however to be associated with an increased risk of lymph node metastasis occurrence (OR = 2.94; P = 0.01). Furthermore, a positive association was found between ApoA1 −75 A allele carriers and breast cancer risk (OR = 1.57; P = 0.02). Regarding prognostic indicators, a significant association was found between ApoA1 (−75) A allele carriers and the premenopausal status of breast cancer patients (OR = 1.73; P = 0.03). Additionally, the presence of the −75 A allele was correlated with the oestrogen receptor status among premenopausal women (OR = 2.45; P = 0.02). This is the first report on the studies of ApoA1 single nucleotide polymorphisms (SNPs) in breast carcinomas. Our data suggest that these genetic variations of ApoA1 may represent a marker for the increased risk of breast cancer.     

Association of MTHFR and PPARγ2 gene polymorphisms in relation to type 2 diabetes mellitus cases among north Indian population

Background

Type 2 diabetes mellitus is a multifactorial and polygenic disease, which is considered as a major life threatening problem all over the world. There has been a worldwide effort in the identification of susceptibility genes for type 2 diabetes mellitus and its complications. At present, adequate data is not available dealing with MTHFR (rs1801133) and PPARγ2 (rs1801282) gene polymorphisms and its association with type 2 diabetes mellitus cases among north Indian populations. Thus, we conceived the need for further studies to investigate MTHFR and PPARγ2 gene polymorphisms and their susceptibility to type 2 diabetes mellitus in north Indian population.

Materials and methods

In this study, a total 175 subjects including 87 type 2 diabetes mellitus cases and 88 controls were enrolled. MTHFR and PPARγ2 gene polymorphisms in the cases and controls were evaluated by polymerase chain reaction and restriction fragment length polymorphism (PCR–RFLP).

Results

The MTHFR gene CC, CT, TT genotype frequencies obtained were 40%, 43%, and 17% in type 2 diabetes mellitus cases and 56%, 29%, and 15% in healthy controls respectively. The OR for CC was 0.54 (95%CI 0.29–0.98, P = 0.041, χ2 = 4.18, power = 0.98), for CT 1.76 (95%CI 0.94–3.30, P = 0.07, χ2 = 3.2, power = 0.96), and for TT 1.2 (95%CI 0.53–2.70, P = 0.66, χ2 = 0.198, power = 0.76). The PPARγ2 gene GG CG, CC genotype frequencies obtained were 28%, 41%, and 31% in cases and 40%, 39%, and 21% in healthy controls respectively. OR for GG was 0.58 (95%CI 0.30–1.09, P = 0.08, χ2 = 2.9, power = 0.96), for CG 1.12 (95%CI 0.61–2.05, P = 0.71, χ2 = 0.137, power = 0.778), and for CC 1.63 (95%CI 0.82–3.23, P = 0.156, χ2 = 2.01, power = 0.92).

Conclusion

It might be recommended that MTHFR CC genotype seems to be a good marker for the early identification of population at risk of type 2 diabetes mellitus. While we have detected significant difference in allelic frequencies of PPARγ2 C (Proline) and G (Alanine), but at genotypic level significant difference was not detected in this case–control study. Further study with larger groups may be required to validate the study.     

Cyclooxygenase-2 −765 G/C polymorphisms and susceptibility to hepatitis B-related liver cancer in Han Chinese population

To investigate the relationship of cyclooxygenase-2 (COX-2) polymorphisms [COX-2 −765 G/C (rs 20417)] and susceptibility to hepatitis B-related liver cancer in Han Chinese population. The polymorphisms of COX-2 −765 G/C was detected by polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) in 300 patients with hepatitis B, 300 patients with cirrhosis, 300 patients with primary liver carcinoma and 300 health controls. The COX-2 −765 G/C genotypes were GG, GC and CC. There frequencies in the hepatitis B patients were 80.33, 17.67 and 2.00%; in the cirrhosis patients were 77.67, 18.00 and 4.33%; in the patients with primary liver carcinoma were 65.67, 28.33 and 6.00% and in the heathy controls were 87.00, 12.33 and 0.67%, respectively, COX-2 −765 C allele carriers had an increased risk of hepatitis B-related liver cancer. COX-2 −765 C allele carriers having drinking history or family history of liver cancer had higher risk for HCC. COX-2 −765 C allele genotype, drinking history and family history of liver cancer may increase the susceptibility to hepatitis B-related liver cancer in Gansu province, China.     

CYP1B1 (4326C > G), CYP2F1 (c.14_15insC), CYP2J2 (?76G > T), and CYP2S1 (13106C > T and 13255A > G) polymorphisms and genetic predisposition to chronic respiratory diseases induced by smoking and occupational factors

The contribution of the polymorphic markers of cytochrome P450 genes to respiratory diseases caused by smoking and occupational factors has been assessed. For this purpose, PCR-RFLP analysis of the CYP1B1 (rs1056836, 4326C > G), CYP2F1 (rs11399890, c.14_15insC), CYP2J2 (rs890293, -76G > T), and CYP2S1 (rs34971233, 13106C > T and rs338583, 13255A > G) gene polymorphisms has been performed. The analysis has shown that CYP1B1 (rs1056836, 4326C > G) and CYP2F1 (rs11399890, c.14_15insC) polymorphisms may contribute to the development of occupational chronic bronchitis. The proportion of CYP1B1*1*3 heterozygotes in the group of patients with occupational chronic bronchitis is considerably greater than in the group of healthy workers (69.16% versus 53.29%; χ² = 5.94, p = 0.02, p _cur = 0.04, OR = 1.97, the 95% CI is 1.13–3.42). Patients with occupational chronic bronchitis and healthy workers significantly differed from each other in the CYP2F1 genotypes frequency distribution (rs11399890, c.14_15insC) (χ² = 6.18, d.f. = 2, p = 0.05). CYP2F1 wild type/ins heterozygous genotype frequency is higher in healthy workers (36.08%) than in patients (22.22%) (χ² = 5.48, p = 0.02, p _cur = 0.04, OR = 0.51, the 95% CI is 0.28–0.90). No association has been found between the CYP2J2 (rs890293, −76G > T) or CYP2S1 (rs34971233, 13106C > T, and rs338583, 13255A > G) gene polymorphisms and respiratory diseases.     

A p.R369G POLG2 mutation associated with adPEO and multiple mtDNA deletions causes decreased affinity between polymerase γ subunits

Human mitochondrial DNA (mtDNA) polymerase γ (pol γ) is the sole enzyme required to replicate and maintain the integrity of the mitochondrial genome. It comprises two subunits, a catalytic p140 subunit and a smaller p55 accessory subunit encoded by the POLG2 gene. We describe the molecular characterization of a potential dominant POLG2 mutation (p.R369G) in a patient with adPEO and multiple mtDNA deletions. Biochemical studies of the recombinant mutant p55 protein showed a reduced affinity to the pol γ p140 subunit, leading to impaired processivity of the holoenzyme complex but did not show sensitivity to N-ethylmalaimide (NEM) inhibition, inferring a novel disease mechanism.     

Association of NADPH oxidase p22phox gene C242T, A640G and −930A/G polymorphisms with primary knee osteoarthritis in the Greek population

Osteoarthritis (OA) is the most common form of arthritis with still unknown pathogenic etiology and considerable contribution of genetic factors. Recently, a new emerging role of oxidative stress in the pathology of OA has been reported, lacking however elucidation of the underlying mechanism. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase being a complex enzyme produced by chondrocytes, presents the major source of reactive oxygen species and main contributor of increased oxidative stress. The present study aims to evaluate the association of NADPH oxidase p22phox gene C242T, A640G and ?A930G polymorphisms with primary knee OA in the Greek population. One hundred fifty five patients with primary symptomatic knee OA participated in the study along with 139 matched controls. Genotypes were determined using polymerase chain reaction and restriction fragment length polymorphism technique. Allelic and genotypic frequencies were compared between both study groups. NADPH p22phox ?A930G polymorphism was significantly associated with knee OA in the crude analysis (P = 0.018). No significant difference was detected for C242T and A640G polymorphisms (P > 0.05). The association between ?A930G polymorphism and knee OA disappeared when the results were adjusted for obesity (P = 0.078, odds ratio 0.54, 95 % CI 0.272–1.071). The interaction between all three polymorphisms was not significant. The present study shows that NADPH oxidase p22phox gene C242T, A640G and ?A930G polymorphisms are not risk factors for knee OA susceptibility in the Greek population. Further studies are needed to give a global view of the importance of this polymorphism in the pathogenesis of OA.     

Three most common nonsynonymous UGT1A6*2 polymorphisms (Thr181Ala,Arg184Serand Ser7Ala) and therapeutic response to deferiprone in β-thalassemia major patients

Deferiprone is used as a chelation agent in chronic iron overload in β-thalassemia patients. Patients on deferiprone therapy show variable response to this drug in terms of reduction in iron overload as well as adverse drug reactions (ADRs). The pharmacogenetic studies on deferiprone have not carried out in patients with blood disorders in India. Therefore, the present study was carried out to evaluate the three most common nonsynonymous UGT1A6 polymorphisms Thr181Ala (541 A/G), Arg184Ser (552 A/C) and Ser7Ala (19 T/G) and therapeutic response to deferiprone in β-thalassemia major patients. Two hundred and eighty six (286) β-thalassemia major patients were involved in the study. Serum ferritin levels were estimated periodically to assess the status of the iron overload and the patients were grouped into responders and non-responders depending on the ferritin levels. The UGT1A6*2 polymorphisms were detected by PCR-RFLP methods. The association between the genotypes and outcome as well as ADRs was evaluated by Open EPI software. A significant difference was observed in the genotypic distribution of UGT1A6*2 Thr181Ala polymorphism in responders and non-responders. However, there was no difference in the genotypic distribution between patients with and without ADRs. As far as the UGT1A6*2 Arg184Ser polymorphism is concerned, no significant difference was observed between responders and non-responders. Further, evaluating the association of UGT1A6*2 Ser7Ala polymorphism with drug response, there was no significant difference in the genotypic distribution between responders and non-responders. However, there was a significant difference between responders with and without ADRs and non-responders with and without ADRs. In addition to this haplotype analysis was also carried out. However, we did not find any specific haplotype to be significantly associated with the deferiprone response in β-thalassemia major patients.     

UCP2 -866G/A,Ala55Val and UCP3 -55C/T Polymorphisms in Association with Obesity Susceptibility — A Meta-Analysis Study

Aims/hypothesis

Variants of UCP2 and UCP3 genes have been reported to be associated with obesity, but the available data on the relationship are inconsistent. A meta-analysis was performed to determine whether there are any associations between the UCP2 -866G/A, Ala55Val, and UCP3 -55C/T polymorphisms and obesity susceptibility.

Methods

The PubMed, Embase, Web of Science and CNKI, CBMdisc databases were searched for all relevant case-control studies. The fixed or random effect pooled measure was determined on the bias of heterogeneity test among studies. Publication bias was examined by the modified Begg''s and Egger''s test.

Results

Twenty-two published articles with thirty-two outcomes were included in the meta-analysis: 12 studies with a total of 7,390 cases and 9,860 controls were analyzed for UCP2 -866G/A polymorphism with obesity, 9 studies with 1,483 cases and 2,067 controls for UCP2 Ala55Val and 8 studies with 2,180 cases and 2,514 controls for UCP3 -55C/T polymorphism. Using an additive model, the UCP2 -866G/A polymorphism showed no significant association with obesity risk in Asians (REM OR = 0.81, 95% CI: 0.65–1.01). In contrast, a statistically significant association was observed in subjects of European descent (FEM OR = 1.06, 95% CI: 1.01–1.12). But neither the UCP2 Ala55Val nor the UCP3 -55C/T polymorphism showed any significant association with obesity risk in either subjects of Asian (REM OR = 0.84, 95% CI: 0.67–1.06 for Ala55Val; REM OR = 0.94, 95% CI: 0.55–1.28 for -55C/T) or of European descent (REM OR = 1.04, 95% CI: 0.80-1.36 for Ala55Val; FEM OR = 1.08, 95% CI: 0.97–1.20 for -55C/T).

Conclusions and Interpretation

Our meta-analysis revealed that the UCP2 -866G/A polymorphism may be a risk factor for susceptibility to obesity in subjects of European descent, but not in individuals of Asian descent. And our results did not support the association between UCP2 Ala55Val, UCP3 -55C/T polymorphisms and obesity in the populations investigated. This conclusion warrants confirmation by further studies.     

The association of polymorphisms in BMP2/MYO1H and skeletal Class II div.1 maxillary and mandibular dimensions. A preliminary ‘report

IntroductionThe genetic impact directly or indirectly predefines maxillofacial dimensions, potentially leading to an inappropriate relationship of the jaws and subsequently skeletal malocclusion. Previous studies focused mainly on genetic polymorphisms and class III malocclusion. This study was set out to investigate the association between genetic polymorphisms in two genes BMP2 (rs235768) and MYO1H (rs11066446) with Class II division 1 malocclusion, skeletal variation in vertical plane, and maxillary and mandibular jaws length.Subjects and methodsSixty patients classified as Skeletal Class I (n = 30) and Class II division 1 (n = 30) were recruited. DNA was extracted from saliva and analyzed by Sanger sequencing. Lateral cephalometric radiographs were measured for the anterio-posterior relationship of maxillary and mandibular arch using digital tracing. Hardy-Weinberg equilibrium analysis of genotype frequencies was performed using Chi-square test to compare genotype distribution among groups and multiple logistic regression analysis adjusted by gender was also performed.ResultsThe rs235768 polymorphism in BMP2 was associated with hypodivergent face, increased maxillary length, and decreased mandibular length. Meanwhile, the rs11066446 polymorphism in MYO1H was associated with decreased mandibular length. New polymorphism was identified in MYO1H (rs10850090) in association with decreased mandibular length.ConclusionA potential association between polymorpisms in BMP2 rs235768 and MOY1H rs11066446 and rs10850090 and Class II division 1 skeletal malocclusion related phenotypes exists, however, the degree of it has to be further investigated and yet to be discovered.     

Association between the −11377 C/G and −11391 G/A polymorphisms of adiponectin gene and adiponectin levels with susceptibility to type 1 and type 2 diabetes mellitus in population from the west of Iran,correlation with lipid profile

Adipose tissue, an endocrine organ, secretes bioactive factors including adiponectin. Adiponectin is a protein hormone that enhances insulin sensitivity through increased fatty acid oxidation and inhibition of hepatic glucose production. We assessed the association of the adiponectin promoter region polymorphisms −11391 G/A and −11377 C/G with susceptibility to type 1 (T1DM) and type 2 (T2DM) diabetes mellitus in the population of west Iran. Also, we investigated the effect of adiponectin level and lipid profile on T1DM and T2DM development. In this case-control study, we recruited 189 patients with diabetes (100 T2DM and 89 T1DM) and 161 sex and age-matched unrelated healthy controls. Adiponectin mutations were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and the protein level was measured by the enzyme-linked immunosorbent assay. Other biochemical parameters were determined by routine laboratory methods. The G allele of adiponectin gene at −11377 position (C/G) significantly increased the risk of T1DM. With respect to genotype models, codominant (2.97 times), dominant (3.6-fold), and over-codominant (2.9-fold) patients with T1DM who carried −11377 C > G single-nucleotide polymorphisms were significantly susceptible to the development of the disease. A significantly higher level of adiponectin in T1DM was oberved compared with the control group. In contrast, patients with T2DM had lower adiponectin levels compared with healthy controls. The genotype distributions of −11391 G/A polymorphisms were the same for patients with diabetes and control groups. The presence of G allele at −11377 C/G adiponectin gene significantly increased serum adiponectin level and may be a risk factor for T1DM susceptibility among the western Iranian population.     

Cleavage-resistant fusion proteins of the M2 muscarinic receptor and Gαi1. Homotropic and heterotropic effects in the binding of ligands

Background

G protein-coupled receptors fused to a Gα-subunit are functionally similar to their unfused counterparts. They offer an intriguing view into the nature of the receptor–G protein complex, but their usefulness depends upon the stability of the fusion.

Methods

Fusion proteins of the M₂ muscarinic receptor and the α-subunit of G_i1 were expressed in CHO and Sf9 cells, extracted in digitonin–cholate, and examined for their binding properties and their electrophoretic mobility on western blots.

Results

Receptor fused to native α_i1 underwent proteolysis near the point of fusion to release a fragment with the mobility of α_i1. The cleavage was prevented by truncation of the α-subunit at position 18. Binding of the agonist oxotremorine-M to the stable fusion protein from Sf9 cells was biphasic, and guanylylimidodiphosphate promoted an apparent interconversion of sites from higher to lower affinity. With receptor from CHO cells, the apparent capacity for N-[³H]methylscopolamine was 60% of that for [³H]quinuclidinylbenzilate; binding at saturating concentrations of the latter was inhibited in a noncompetitive manner at low concentrations of unlabeled N-methylscopolamine.

Conclusions

A stable fusion protein of the M₂ receptor and truncated α_i1 resembles the native receptor–G protein complex with respect to the guanyl nucleotide-sensitive binding of agonists and the noncompetitive binding of antagonists.

General significance

Release of the α-subunit is likely to occur with other such fusion proteins, rendering the data ambiguous or misleading. The properties of a chemically stable fusion protein support the notion that signaling proceeds via a stable multimeric complex of receptor and G protein.     

α-Thalassemia Associated with Hb Instability: A Tale of Two Features. The Case of Hb Rogliano or α1 Cod 108(G15)Thr→Asn and Hb Policoro or α2 Cod 124(H7)Ser→Pro.

We identified two new variants in the third exon of the α-globin gene in families from southern Italy: the Hb Rogliano, α1 cod108 ACC>AAC or α1[α108(G15)Thr→Asn] and the Hb Policoro, α2 cod124 TCC>CCC or α2[α124(H7)Ser→Pro]. The carriers showed mild α-thalassemia phenotype and abnormal hemoglobin stability features. These mutations occurred in the G and H helices of the α-globin both involved in the specific recognition of AHSP and β1 chain. Molecular characterization of mRNA, globin chain analyses and molecular modelling studies were carried out to highlight the mechanisms causing the α-thalassemia phenotype. The results demonstrated that the α-thalassemia defect associated with the two Hb variants originated by different defects. Hb Rogliano showed an intrinsic instability of the tetramer due to anomalous intra- and inter-chain interactions suggesting that the variant chain is normally synthesized and complexed with AHSP but rapidly degraded because it is unable to form the α1β1 dimers. On the contrary in the case of Hb Policoro two different molecular mechanisms were shown: the reduction of the variant mRNA level by an unclear mechanism and the protein instability due to impairment of AHSP interaction. These data highlighted that multiple approaches, including mRNA quantification, are needed to properly identify the mechanisms leading to the α-thalassemia defect. Elucidation of the specific mechanism leads to the definition of a given phenotype providing important guidance for the diagnosis of unstable variants.     

The Saccharomyces cerevisiae protein YJR043C (Pol32) interacts with the catalytic subunit of DNA polymerase α and is required for cell cycle progression in G2?/?M

We have analysed the YJR043c gene of Saccharomyces cerevisiae, previously identified by systematic sequencing. The deletion mutant (yjr043cΔ) shows slow growth at low temperature (15° C), while at 30° C and 37° C the growth rate of mutant cells is only moderately affected. At permissive and nonpermissive temperatures, mutant cells were larger and showed a high proportion of large-budded cells with a single duplicated nucleus at or beyond the bud neck and a short spindle. This phenotype was even more striking at low temperature, the mutant cells becoming dumbbell shaped. All these phenotypes suggest a role for YJR043C in cell cycle progression in G2/M phase. In two-hybrid assays, the YJR043c gene product specifically interacted with Poll, the catalytic subunit of DNA polymerase α. The pol1-1 /yjr043cΔ double mutant showed a more severe growth defect than the pol1-1 single mutant at permissive temperature. Centromeric plasmid loss rate elevated in yjr043cΔ. Analysis of the sequence upstream of the YJR043c ORF revealed the presence of an MluI motif (ACGCGT), a sequence associated with many genes involved in DNA replication in budding yeast. The cell cycle phenotype of the yjr043cΔ mutant, the evidence for genetic interaction with Pol1, the presence of an MluI motif upstream and the elevated rate of CEN plasmid loss in mutants all support a function for YJR043C in DNA replication. Received: 22 July 1998 / Accepted: 22 September 1998     

Stimulation of hexose uptake in rat thymic lymphocytes by phorbol ester. A role for Ca2+ and Na+H+ exchange?

The tumor promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA) stimulates hexose uptake into rat thymocytes. This study explores two possible messengers of this stimulation: changes in cytosolic [Ca2+], and activation of the Na+/H+ antiport. The cytosolic level of Ca2+, determined by the fluorescence of quin-2, was elevated by TPA, and this rise required extracellular Ca2+. In contrast, stimulation of hexose uptake was still observed in Ca2+ -free media even when cytoplasmic [Ca2+] was buffered with quin-2. TPA also raised the cytoplasmic pH, presumably through activation of the Na+/H+ exchange. However, replacement of extracellular Na+ by N-methylglucamine+ or choline+ which prevents the cytoplasmic alkanization did not prevent stimulation of hexose uptake by TPA. Moreover, amiloride, at concentrations that inhibit Na+/H+ exchange in these cells, did not interfere with stimulation of hexose uptake by TPA. In conclusion, stimulation of hexose uptake by phorbol ester in rat thymocytes does not appear to be mediated by changes in cytosolic free Ca2+ or in the activity of the Na+/H+ antiport.