Renal function in relation to three candidate genes in a Chinese population

We recently found in a white population that the genes encoding angiotensin-converting enzyme (ACE, I/D polymorphism), rc="/content/0vduy45exeeuw7r8/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-adducin (Gly460Trp), and aldosterone synthase (–344C/T) jointly influence renal function. We therefore investigated in a Chinese population the associations between the serum concentrations of creatinine and uric acid and these three genetic polymorphisms. We genotyped 471 ethnic Han Chinese subjects from 125 nuclear families recruited in northern China via random population sampling (75%) and at specialized hypertension clinics (25%). We performed population-based and family-based association analyses using generalized estimating equations (GEE) and quantitative transmission disequilibrium test (QTDT), respectively, while controlling for covariables. The participants were 39.7 years old and included 235 women (49.9%). The blood pressure measured at the subjectsrc="/content/0vduy45exeeuw7r8/xxlarge8217.gif" alt="rsquo" align="BASELINE" BORDER="0"> homes averaged 126/80 mmHg. Mean values were 71 µmol/l for serum creatinine, 111 ml min^–1 1.73 m^–2 for calculated creatinine clearance, and 236 µmol/l for serum uric acid. With adjustment for covariables, GEE analyses of single genes demonstrated that serum uric acid, but not serum creatinine, was positively associated with the ACE D allele. Serum uric acid concentrations were 15.8 µmol/l (95% confidence interval 3.3–28.2) and 25.7 µmol/l (11.1–40.2) higher in DD homozygotes than in ID and II subjects, respectively. Further GEE analyses of the three genes combined showed that the association between serum uric acid and the ACE polymorphism was confined to carriers of the rc="/content/0vduy45exeeuw7r8/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-adducin Gly and/or aldosterone synthase C alleles. Sensitivity analyses in parents and offspring separately as well as QTDT analyses were confirmatory. Among 114 informative offspring carrying the rc="/content/0vduy45exeeuw7r8/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-adducin Gly allele serum uric acid was significantly and positively associated with the transmission of the ACE D allele (rc="/content/0vduy45exeeuw7r8/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">=20.7 µmol/l). In conclusion, the present study extends our previous findings on the combined effects of the three candidate genes and supports the concept that these genetic polymorphisms jointly influence renal function.     

Amino-acid alterations in the β-tubilin gene of Neurospora crassa that confer resistance to carbendazim and diethofencarb

We have previously shown that increased sensitivity to diethofencarb in the carbendazim(MBC)-resistant F914 strain of Neurospora crassa is caused by a single amino-acid change in rc="/content/r0x2178555h70508/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-tubulin, ¹⁹⁸Glu to Gly. Three diethofencarb-resistant mutants that are also resistant to MBC were isolated from strain F914. They contained single base-pair-substitution mutations in the rc="/content/r0x2178555h70508/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-tubulin gene. The amino acid changes in rc="/content/r0x2178555h70508/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-tubulin, Phe from ²⁵⁰Leu, Val from ¹⁶⁵Ala, and Ala from ²³⁷Thr, were responsible for diethofencarb-resistance in the mutant strains FR511, FR513, and FR421, respectively. The amino acid at position 198 of rc="/content/r0x2178555h70508/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-tubulin in these mutants was Gly, which is the same as in strain F914. rc="/content/r0x2178555h70508/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-tubulin genes with ¹⁹⁸Glu were constructed by site-directed mutagenesis. The altered rc="/content/r0x2178555h70508/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-tubulin genes derived from FR511 and FR421 transformed the wild-type strain to resistance to MBC, indicating that ²⁵⁰Phe and ²³⁷Ala in rc="/content/r0x2178555h70508/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-tubulin are responsible for resistance not only to diethofencarb but also to MBC.     

Outcome of surgical treatment for early adenocarcinoma of the esophagus or gastro-esophageal junction

Adenocarcinoma of the esophagus, or GEJ, has a poor prognosis. Early lesions [i.e. high grade dysplasia (HGD) or T1-carcinoma] are potentially curable. Local endoscopic therapies are promising treatment options for superficial lesions; however, for deeper lesions, surgical resection is considered to be the treatment of choice. To contribute to therapeutic decision-making, we retrospectively analysed the outcome of transhiatal esophagectomy in 120 patients with pathologically proven HGD (n=13) or T1-adenocarcinoma (n=107) of the distal esophagus or gastro-esophageal junction (GEJ). Tumors were subdivided into six different depths of invasion (rc="/content/m4p55m136671131r/xxlarge8216.gif" alt="lsquo" align="BASELINE" BORDER="0">T1-mucosalrc="/content/m4p55m136671131r/xxlarge8217.gif" alt="rsquo" align="BASELINE" BORDER="0"> m1-m3, rc="/content/m4p55m136671131r/xxlarge8216.gif" alt="lsquo" align="BASELINE" BORDER="0">T1-submucosalrc="/content/m4p55m136671131r/xxlarge8217.gif" alt="rsquo" align="BASELINE" BORDER="0"> sm1-sm3), and the frequency of lymphatic dissemination and time to locoregional and/or distant recurrence were analysed. Only one of the 79 T1m1-3/sm1 tumors (1%) showed lymph node metastases as compared with 18 out of 41 T1sm2-3 tumors (44%). There was a significant difference in recurrence-free period between T1m1-m3/sm1 versus T1sm2-sm3 tumor patients (P log rank <0.0001), with 5-year recurrence-free percentages of 97% and 57%, respectively. In multivariate analysis including age, gender, tumor differentiation grade, N-stage and depth of invasion, only N-stage was an independent prognostic factor for recurrence-free period (hazard rate=5.9, 95% CI 1.7–20.7). However, if N-stage was excluded from analysis, only depth of invasion (T1sm2-3 versus T1m1-m3/sm1) was an independent prognostic factor for recurrence-free period (hazard rate=7.5, 95% CI 2.0–27.7). These data indicate that T1m1-m3/sm1 adenocarcinomas of esophagus or GEJ show a very low risk of lymphatic dissemination and are therefore eligible for local endoscopic therapy. After transhiatal surgical resection, almost half of the patients with T1sm2-sm3 lesions develop recurrent disease within 5 years, and therefore need additional therapy to improve survival.     

Interferon-α activates cytotoxic function but inhibits interleukin-2-mediated proliferation and tumor necrosis factor-α secretion by immature human natural killer cells

Natural killer (NK) cells play an important role in host defense mechanisms against infection and neoplasia. Interferon-rc="/content/r48l81278174430m/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> (IFN-rc="/content/r48l81278174430m/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">) has been shown to activate NK cells and to augment their cytotoxic activity, albeit its role in the maturation pathway of NK cells has not been elucidated. The present study examined whether IFN-rc="/content/r48l81278174430m/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> activates the immature NK subset (Free cells) to become cytotoxic and also ascertained whether IFN-rc="/content/r48l81278174430m/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> uses the same pathway of activation as that mediated by interleukin-2 (IL-2). Incubation of sorted Free cells overnight with IFN-rc="/content/r48l81278174430m/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> resulted in augmentation of their cytotoxic function against NK sensitive target cells. The enhanced cytotoxic activity was not accompanied by a new recruitment of NK-target binder cells but by an increase in the frequency of killer cells in the conjugate fraction. Activation of the Free subset by IFN-rc="/content/r48l81278174430m/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> resulted in upregulation of CD69, CD11b, and CD2 surface expression and stimulated secretion of IFN-rc="/content/r48l81278174430m/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">. Unlike IL-2, IFN-rc="/content/r48l81278174430m/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> did not stimulate the Free cells to proliferate or secrete TNF-rc="/content/r48l81278174430m/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> and activation of cytotoxicity and modulation of surface antigens by IFN-rc="/content/r48l81278174430m/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> were independent of TNF-rc="/content/r48l81278174430m/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">. The failure of IFN-rc="/content/r48l81278174430m/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> to stimulate secretion and proliferation by Free cells appeared to be mediated by negative signals. This was corroborated in experiments demonstrating that when Free cells were cultured with both IFN-rc="/content/r48l81278174430m/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> and IL-2, a significant inhibition was observed for both the IL-2 dependent secretion of TNF-rc="/content/r48l81278174430m/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> and proliferation. These results demonstrate that IFN-rc="/content/r48l81278174430m/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> serves as both an activator and a regulator of NK function. Further, activation of the immature Free NK cells by IL-2 and IFN-rc="/content/r48l81278174430m/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> proceeds by TNF-rc="/content/r48l81278174430m/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-dependent and independent pathways, respectively. The findings also support our contention that the mechanism of activation of the cytotoxic machinery of NK cells is not linked to the mechanism of activation of cytokine secretion and/or proliferation.Abbreviations used IFN interferon - IL interleukin - PBL peripheral blood leukocytes - PE phycoerythrin - PE-GAM PE-conjugated Fabrc="/content/r48l81278174430m/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">2 goat anti-mouse IgG - NK natural killer - NRS normal rabbit serum - TNF tumor necrosis factor - FCS fetal calf serum - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - MACS magnetic cell sorting - ELISA enzyme-linked immunosorbent assay - BSA bovine serum albumin - PKC protein kinase C - mAb monoclonal antibody - PBMC peripheral blood mononuclear cells - BCLL B-chronic lymphocytic leukemia - E effector - T target     

Alteration of regulatory enzyme activities in fast-twitch and slow-twitch muscles and muscle fibres in low-intensity endurance-trained rats

The effect of progressive, low-intensity endurance training on regulatory enzyme activities in slow-twitch (ST) and fast-twitch (FT) muscle fibres was studied in 32 rats. Of those rats 16 were trained on a treadmill at a running speed of 10m · min^–1 5 days a week over an 8-week period. Running time was progressively increased from 15 min to 2 h · day^–1. Of the rats 4 trained and 4 sedentary rats were also subjected to acute exhausting exercise. Enzyme activities of phosphofructokinase 1 (PFKI) from glycolysis, rc="/content/m42r16l6841p0330/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-ketoglutarate dehydrogenase (rc="/content/m42r16l6841p0330/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-KGDH) from the Krebs cycle and carnitine palmitoyltransferase (CPT I and II) from fatty acid metabolism in soleus, tibialis anterior and gastrocnemius muscles were measured in trained and sedentary rats. Enzyme activities of individual ST and FT fibres were measured from the freeze-dried gastrocnemius muscle of 8 trained and 8 sedentary rats. In the sedentary rats the activity of PFK1 in tibialis anterior and soleus muscles was 141% and 41% of the activity in gastrocnemius muscle, respectively. The activity of rc="/content/m42r16l6841p0330/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-KGDH in tibialis anterior and soleus muscles was 164% and 278% of the activity in gastrocnemius muscle, respectively. The activity of CPT I in tibialis anterior and gastrocnemius muscles were at the same level, but in soleus muscle the activity was 127% of that in mixed muscle. Endurance training increased enzyme activities of rc="/content/m42r16l6841p0330/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-KGDH and CPT I significantly (P < 0.05) in gastrocnemius muscle but not in soleus or tibialis anterior muscle. After training both rc="/content/m42r16l6841p0330/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-KGDH and CPT II activities were elevated significantly (P < 0.05) in the ST fibres of gastrocnemius muscle, whereas in FT fibres only rc="/content/m42r16l6841p0330/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-KGDH was increased. For PFK1 activity no significant change was observed in ST or FT fibres. After acute exercise, activities of mitochondrial enzymes rc="/content/m42r16l6841p0330/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-KGDH and CPT I tended to be elevated in all muscles. Thus, low-intensity endurance training induced significant peripheral changes in regulatory enzyme activities in oxidative and fatty acid metabolism in individual ST or FT muscle fibres.     

Two phylogenetically highly distinct β-tubulin genes of the basidiomycete Suillus bovinus

Genes tubb1 and tubb2 which encode beta-tubulins 1 and 2, respectively, were characterised from the ectomycorrhizal basidiomycete Suillus bovinus. The two beta-tubulins are surprisingly divergent, with the lowest known sequence identity (60%) in any single fungal species. Comparative analysis showed that beta-tubulin 1 and the intron distribution within the tubb1 gene resemble the other beta-tubulins. beta-Tubulin 2, in contrast, is the most divergent fully described fungal beta-tubulin and the gene contains at least 21 introns, which is the largest amount known for any beta-tubulin gene. Despite this divergence, both genes are constitutively expressed in the functional compartments of the mycorrhizosphere and in pure cultures. Transcription of tubb1 is about 2.4 times higher than that of tubb2; and this difference is also seen at the translation level. Evidence suggested that phosphorylation may be the main post-translational modification of both beta-tubulins. The putative GTP-binding site residues of beta-tubulin 1 match crystallised pig beta-tubulin residues, while five of the nine differences in beta-tubulin 2 match the pig alpha-tubulin GTP-site, suggesting the presence of adaptive sequence evolution. In a Bayesian analysis, beta-tubulin 1 joins the other basidiomycete sequences, while beta-tubulin 2 loosely associates with the group of divergent ascomycete sequences without any clear relative among the known full-length fungal beta-tubulin sequences.     

The structuring of an allergy index based on IgE-mediated skin sensitivity to common environmental allergens

We computed skin-test sensitivity levels in 485 adults puncture-tested with eight standardized, high-quality inhalant allergens tested at single concentrations. In order to quantitate the "average" IgE-mediated skin sensitivity of each subject, we used both nonparametric and parametric statistical methods to generate two "allergy indices" (Allergy Index I and Allergy Index II) based on sensitivity end-point data from the subpopulations of individuals positive to six of the eight allergens. For the 192 skin test-positive subjects, Allergy Index I and Allergy Index II were significantly correlated with each other (rs = 0.98, p less than 0.001) and with the number of positive skin-test reactions (rs congruent to 0.9, p less than 0.001) as well as with log[total serum IgE] (r congruent to 0.4, p less than 0.01). In 102 ragweed-positive subjects, log[specific IgE to ragweed] was significantly correlated with ragweed-specific "ragweed indices I and II" (r congruent to 0.6, p less than 0.01). Furthermore, the average daily symptom scores reported by 14 ragweed-positive subjects during the ragweed pollination season were significantly correlated with ragweed indices I and II (p less than 0.05). We propose the use of Allergy Index II in epidemiologic and genetic studies of allergic phenotypes as well as in clinical decisions for diagnosis and immunotherapeutic intervention.     

Isoenzymes of γ-glutamyl transpeptidase in liver diseases

Summary A new method for the separation of isoenzymes ofrc="/content/q6471173jl0r5j00/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">-glutamyl-transpeptidase is described, using electrophoresis on acetate cellulose gel and a developing solution composed byrc="/content/q6471173jl0r5j00/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">-glutamylrc="/content/q6471173jl0r5j00/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-naphthylamide, and a colored diazonium compound.The method permits the separation of up to four different isoenzymes, which we calledrc="/content/q6471173jl0r5j00/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">-GT₁,rc="/content/q6471173jl0r5j00/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">-GT₂,rc="/content/q6471173jl0r5j00/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">-GT₃,rc="/content/q6471173jl0r5j00/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">-GT₄, the first two showing an electrophoretic migration similar to that ofrc="/content/q6471173jl0r5j00/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> ₁- andrc="/content/q6471173jl0r5j00/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> ₂-globulins and the other two to that ofrc="/content/q6471173jl0r5j00/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-globulins.The present technique has proved its usefulness in detecting isoenzymes in serum with values of totalrc="/content/q6471173jl0r5j00/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">-glutamyl-transpeptidase higher than 80 U/L.The application of this method in 52 patients with different types of biliary obstruction and hepatocellular damage has shown that it provides new possibilities in differential diagnosis.     

An investigation of depotentiation of long-term potentiation in the CA1 region of the hippocampus

We have investigated long-term synaptic depression in the CA1 region of rat hippocampal slices. Prolonged low-frequency stimulation (LFS; 900 stimuli delivered at 2 Hz) of the Schaffer collateral-commissural pathway in naïve slices did not induce long-term depression (LTD) of synaptic transmission. However, if long-term potentiation (LTP) was firstly induced in the pathway then LFS generated an LTD-like effect (i.e. depotentiation of LTP). Depotentiation could be induced 2 h (the longest time studied) after the induction of LTP and was stable for the duration of the experiment (followed for up to 40 min). The induction of depotentiation was not blocked by the N-methyl-d-aspartate receptor antagonist d-2-amino-5-phosphonopentanoate, the L-type voltage-gated Ca²⁺ channel blocker nimodipine or the nitric oxide synthase inhibitor Nrc="/content/r7108p8531282j78/xxlarge969.gif" alt="ohgr" align="BASELINE" BORDER="0">-nitro-l-arginine. However, the magnitude of depotentiation was reversibly reduced, in a stereoselective manner, by the specific metabotropic glutamate receptor (mGluR) antagonist (+)-rc="/content/r7108p8531282j78/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-methyl-4-carboxyphenylglycine. These results show that prolonged low frequency stimulation can result in an mGluR-dependent depotentiation of LTP.     

Identification of a functional mutation in pp32r1 (ANP32C)

No mutations or polymorphisms have previously been reported in pp32r1 (ANP32C; GenBank: AF008216.1). pp32r1 is part of the highly conserved ANP32 family, some of whose members are associated with control of histone acetylation, mRNA stability, and specialized forms of apoptosis. Although 87.6% identical at the protein level, pp32r1 is functionally distinct from pp32 (ANP32A) in its failure to suppress oncogenesis in in vitro transformation systems and its tumorigenicity in in vivo assays. The present study found that pp32r1 expression levels vary among human tumor cell lines, with the highest levels found in prostatic adenocarcinoma cell lines. pp32r1 also appears to be polymorphic at nucleotide g.4520 and nucleotide g.4664 in human tobacco-associated oral mucosal lesions, human fibroblast cell lines, and several carcinoma cell lines. PC-3 human prostatic adenocarcinoma cells likewise appear to be polymorphic at these loci, but additionally contain a g.4870T>C transversion mutation. The mutation results in a p.Tyr140His substitution, which lies in a functionally important region of the molecule. In the PC-3 prostate cancer line, the mutation is either homozygous, or hemizygous accompanied by loss of heterozygosity. ACHN cells stably transfected with pp32r1 containing this mutation showed a markedly increased rate of growth. The pp32r1 mutation could thus be causally associated with the neoplastic growth properties of PC-3, and be of potential clinical significance.