Expression of the Androgen Receptor,pAkt, and pPTEN in Breast Cancer and Their Potential in Prognostication

BACKGROUNDImportance of androgen receptor (AR) as an independent prognostic marker in Pakistani women with breast cancer (BCa) remains unexplored. Our aim was to identify the expression and potential prognostic value of AR, its upstream regulator (pAkt) and target gene (pPTEN) in invasive BCa.METHODSThis study used a cohort of 200 Pakistani women with invasive BCa diagnosed during 2002-2011. Expression of AR, pAkt and pPTEN was determined on formalin fixed paraffin embedded tissue sections by immunohistochemistry. The association of AR, pAkt and pPTEN with clinicopathological parameters was determined. Survival analyses were undertaken on patients with ≥ 5 years of follow-up (n = 82).RESULTSExpression of AR, pAkt and pPTEN was observed in 47.5%, 81.3% and 50.6% of patients, respectively. AR-expressing tumors were low or intermediate in grade (P < .001) and expressed ER (P = .002) and PR (P = .001). Patients with AR⁺ tumors had significantly higher OS (Mean OS = 10.2 ± 0.465 years) compared to patients with AR^? tumors (Mean OS = 5.8 ± 0.348 years) (P = .047). Furthermore, AR-positivity was associated with improved OS in patients receiving endocrine therapy (P = .020). Patients with AR⁺ /pAkt⁺ /pPTEN^? tumors, had increased OS (Mean OS = 7.1 ± 0.535 years) compared to patients with AR^?/pAkt⁺/pPTEN^? tumors (Mean OS = 5.1 ± 0.738 years).CONCLUSIONAR-expressing tumors are frequently characterized by low or intermediate grade tumors, expressing ER and PR. In addition, expression of AR, pAkt and pPTEN, could be considered in prognostication of patients with invasive BCa.     

A hydrophobic sequence at position 313-316 (Leu-Ala-Phe-Trp) in the fifth domain of apolipoprotein H (beta2-glycoprotein I) is crucial for cardiolipin binding.

Apolipoprotein H (apoH, protein; APOH, gene) binds to negatively charged phospholipids, which triggers the production of a subset of autoantibodies against phospholipid in patients with autoimmune diseases. We have demonstrated that two naturally occurring missense mutations in the fifth domain of apoH, Trp316Ser and Cys306Gly, disrupt the binding of native apoH to phosphatidylserine [Sanghera, D. K., Wagenknecht, D. R., McIntyre, J. A. & Kamboh, M. I. (1997) Hum. Mol. Genet. 6, 311-316]. To confirm whether these are functional mutations, we mutagenized APOH cDNAs and transiently expressed them in COS-1 cells. The cardiolipin ELISA of wild-type and mutant recombinant apoH confirmed that the Gly306 and Ser316 mutations are responsible for abolishing the binding of recombinant apoH to cardiolipin. These mutations, however, had no effect on the levels of expression or secretion of recombinant apoH in transfected COS-1 cells. While the Cys306Gly mutation disrupts a disulfide bond between Cys306 and Cys281, which appears to be critical for clustering positively charged amino acids, the Trp316Ser mutation affects the integrity of an evolutionarily conserved hydrophobic sequence at position 313-316 (Leu-Ala-Phe-Trp), which is hypothesized to interact with anionic phospholipid. To test this hypothesis, we exchanged the remaining three hydrophobic amino acids with neutral amino acids by site-directed mutagenesis (Leu313Gly, Ala314Ser and Phe315Ser). Binding of the Leu313Gly and Phe315Ser mutants to cardiolipin was significantly reduced to 25% and 13%, respectively, of that of the wild-type. On the other hand, the Ala314Ser mutation showed normal cardiolipin binding. Taken together with our previous findings, these results strongly suggest that the configuration of the fifth domain of apoH, as well as the integrity of the highly conserved hydrophobic amino acids at positions 313-316, is essential for the binding of apoH to anionic phospholipid.     

Synaptotagmin II and Gangliosides Bind Independently with Botulinum Neurotoxin B but Each Restrains the Other

Botulinum neurotoxin type B (BoNT/B) initiates its toxicity by binding to synaptotagmin II (SytII) and gangliosides GD1a and GT1b on the neural membrane. We synthesized two 27-residue peptides that carry the BoNT/B binding sites on mouse SytII (mSytII 37–63) or human SytII (hSytII 34–60). BoNT/B bound to these peptides, but showed substantially higher binding to mSytII peptide than to hSytII peptide. The mSytII peptide inhibited almost completely BoNT/B binding to synaptosomes (snps) and displayed a high affinity. BoNT/B bound strongly to mSytII peptide and binding was inhibited by the peptide. Binding of BoNT/B to snps was also inhibited (~80 %) by a larger excess of gangliosides GD1a or GT1b. The mSytII peptide inhibited very strongly (at least 80 %) the toxin binding to snps, while the two gangliosides were much less efficient inhibitors requiring much larger excess to achieve similar inhibition levels. Furthermore, gangliosides GD1a or GT1b inhibited BoNT/B binding to mSytII peptide at a much larger excess than the inhibition by mSytII peptide. Conversely, BoNT/B bound well to each ganglioside and binding could be inhibited by the correlate ganglioside and much less efficiently by the mSytII peptide. There was no apparent collaboration between mSytII peptide and either ganglioside. mSytII peptide displayed some protective activity in vivo in mice against a lethal BoNT/B dose. We concluded that SytII peptide and gangliosides bind independently but, with their binding sites on BoNT/B being spatially close, each can influence BoNT/B binding to the other due to regional conformational perturbations or steric interference or both. Ganglioside involvement in BoNT/B binding might help in toxin translocation and endocytosis.     

Two closely linked tomato HKT coding genes are positional candidates for the major tomato QTL involved in Na+/K+ homeostasis

The location of major quantitative trait loci (QTL) contributing to stem and leaf [Na⁺] and [K⁺] was previously reported in chromosome 7 using two connected populations of recombinant inbred lines (RILs) of tomato. HKT1;1 and HKT1;2, two tomato Na⁺‐selective class I‐HKT transporters, were found to be closely linked, where the maximum logarithm of odds (LOD) score for these QTLs located. When a chromosome 7 linkage map based on 278 single‐nucleotide polymorphisms (SNPs) was used, the maximum LOD score position was only 35 kb from HKT1;1 and HKT1;2. Their expression patterns and phenotypic effects were further investigated in two near‐isogenic lines (NILs): 157‐14 (double homozygote for the cheesmaniae alleles) and 157‐17 (double homozygote for the lycopersicum alleles). The expression pattern for the HKT1;1 and HKT1;2 alleles was complex, possibly because of differences in their promoter sequences. High salinity had very little effect on root dry and fresh weight and consequently on the plant dry weight of NIL 157‐14 in comparison with 157‐17. A significant difference between NILs was also found for [K⁺] and the [Na⁺]/[K⁺] ratio in leaf and stem but not for [Na⁺] arising a disagreement with the corresponding RIL population. Their association with leaf [Na⁺] and salt tolerance in tomato is also discussed.     

Protein structure probed by polarization spectroscopy. II. A time-resolved fluorescence study of human fibrinogen

Human fibrinogen in solution was studied by monitoring the time-resolved depolarization of the fluorescence emitted by two spectroscopic labels of which the fluorescence lifetimes differ by an order of magnitude. Contrary to a long-held view, no evidence of molecular flexibility was found in the 10-1000 ns range. In addition, from the rate of the overall rotation, it is proposed that a prolate and symmetric ellipsoid of 47 X 10.5 nm may represent the time-averaged hydrodynamic size and shape of the protein in solution. This rigid and highly hydrated structure (4 g water/g protein) accommodates the latest nodular models obtained from electron microscopy, explains the singular hydrodynamics of fibrinogen and, apparently, it would perform the two main functions of the protein in haemostasis, blood coagulation and platelet aggregation, more efficiently than the flexible molecule.     

Promoter diversity in multigene transformation

Multigene transformation (MGT) is becoming routine in plant biotechnology as researchers seek to generate more complex and ambitious phenotypes in transgenic plants. Every nuclear transgene requires its own promoter, so when coordinated expression is required, the introduction of multiple genes leads inevitably to two opposing strategies: different promoters may be used for each transgene, or the same promoter may be used over and over again. In the former case, there may be a shortage of different promoters with matching activities, but repetitious promoter use may in some cases have a negative impact on transgene stability and expression. Using illustrative case studies, we discuss promoter deployment strategies in transgenic plants that increase the likelihood of successful and stable multiple transgene expression.     

Identification of RAPD markers linked to a major blast resistance gene in rice

Rice blast, caused byPyricularia grisea, is a major production constraint in many parts of the world. The Korean rice variety Tongil showed high levels of resistance for about six years when widely planted under highly disease-conducive conditions, before becoming susceptible. Tongil was found to carry a single dominant gene, designatedPi-10t, conferring resistance to isolate 106 of the blast pathogen from the Philippines. We report here the use of bulked segregant RAPD analysis for rapid identification of DNA markers linked toPi-10t. Pooled DNA extracts from five homozygous blast-resistant (RR) and five susceptible (rr) BC₃F₂ plants, derived from a CO39 × Tongil cross, were analyzed by RFLP using 83 polymorphic probes and by RAPD using 468 random oligomers. We identified two RAPD markers linked to thePi-10t locus: RRF6 (3.8 ± 1.2 cM) and RRH18 (2.9 ± 0.9 cM). Linkage of these markers withPi-10t was verified using an F2 population segregating forPi-10t. The two linked RAPD markers mapped 7 cM apart on chromosome 5. Chromosomal regions surrounding thePi-10t gene were examined with additional RFLP markers to define the segment introgressed from the donor genome.Pi-10t is likely to be a new blast-resistance locus, because no other known resistance gene has been mapped on chromosome 5. These tightly linked RAPD markers could facilitate early selection of thePi-10t locus in rice breeding programmes.     

Role of abscisic acid in regulation of wheat seedling growth under salinity stress

Growth of wheat seedlings (Triticum aestivum L. cv. Mehran-89), in hydroponic culture, was affected by abscisic acid (ABA). Using salinity stress and exogenous ABA application (10-6 M) to enhance endogenous ABA level, the growth of roots was more suppressed than the growth of shoots. On the other hand, norflurazon, which inhibits ABA biosynthesis, reduced only the growth of shoots. This revised version was published online in July 2006 with corrections to the Cover Date.