Nuclear location of hormone-free estrogen receptors by monoclonal antibodies could be a tissue-fixation dependent artifact

The 'two-step' model proposed by Jensen and his collaborators for explaining estrogen action conceptualized hormone-free estrogen receptors (ER) to be cytoplasmic, and hormone-filled, transformed ER to be nuclear. Applying monoclonal antibodies which recognized epitopes in ER and formaldehyde-fixed tissues, King et al demonstrated exclusively nuclear staining in target tissues utilizing immunoperoxidase technique. Recently these antibodies have become commercially available enabling other investigators to conduct studies. In this report, using these monoclonal antibodies we have demonstrated that a change in the concentration of formaldehyde alters the staining pattern yielding cytoplasmic instead of nuclear staining in calf uterus, MCF-7 cells, and ER(+) human breast cancer. In addition, neutralization of the antibody activity was not achieved with freshly prepared ER(+) cytosols. Formaldehyde-treated cytosols were essential. These results ought to caution investigators in determining in vivo location of antigens based on the staining pattern obtained in fixed tissues. Furthermore, this effect of formaldehyde on estrogen receptors may be applicable to other steroid hormone receptors.     

The rate and pattern of nucleotide substitution in Drosophila mitochondrial DNA.

The nucleotide sequences of a segment of mitochondrial DNA (mtDNA) have been determined for nine species or subspecies of the subgenus Drosophila of the genus Drosophila. This segment contains two complete protein-coding genes (i.e., NADH dehydrogenase subunit 1 and cytochrome b) and a transfer RNA gene (tRNA(ser)). The G+C content at third-codon positions for the two protein-coding genes was 1.5 times higher than that in the D. melanogaster species group, which belongs to the subgenus Sophophora. However, there was a substantial difference between the nucleotide frequencies of G and C. The number of nucleotide substitutions per silent site was more than three times higher than that for nuclear DNA, although it was only 60% of that for mammalian mtDNA. Both parametric and nonparametric analyses revealed a strong transition-transversion bias in nucleotide substitution, as was observed in mammalian mtDNA. Moreover, the rate of substitution of A and T for G and C is higher than that for the opposite direction. This bias seems to be responsible for the extremely A+T-rich base composition of Drosophila mtDNA. It is also noted that the rate of transitional change between A and G is higher than that between T and C.     

Cloning of the alpha-amylase cDNA of Aspergillus shirousamii and its expression in Saccharomyces cerevisiae.

alpha-Amylase cDNA was cloned and sequenced from Aspergillus shirousamii RIB2504. The putative protein deduced from the cDNA open reading frame (ORF) consisted of 499 amino acids with a molecular weight of 55,000. The amino acid sequence was identical to that of the ORF of the Taka-amylase A gene of Aspergillus oryzae, while the nucleotide sequence was different at two and six positions in the cDNA ORF and 3' non-coding regions, respectively, so far determined. The alpha-amylase cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast ADH1 promoter using a YEp-type plasmid, pYcDE1. The cDNA of glucoamylase, which was previously cloned from the same organism, was also expressed under the same conditions. Consequently, active alpha-amylase and glucoamylase were efficiently secreted into the culture medium. The amino acid sequence of the N-terminal regions of these enzymes purified from the yeast culture medium confirmed that the signal sequences of these enzymes were cleaved off at the same positions as those of the native enzymes of A. shirousamii.     

Deletion analysis of the Taka-amylase A gene promoter using a homologous transformation system in Aspergillus oryzae.

The Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The molecular mechanism of the induction was investigated using a fusion of the amyB promoter and the Escherichia coli uidA gene encoding beta-glucuronidase (GUS). To identify the region responsible for high-level expression and regulation within the amyB promoter, a series of deletion promoters was constructed and introduced into the A. oryzae met locus by homologous recombination. Deletion of the region between -377 to -290 (the number indicates the distance in base pairs from the translation initiation point (+1) to the deletion end point) significantly reduced of the GUS activity, but slight reduction of the GUS activity was observed in deletions up to -377. Northern blot analysis showed that reduction of the GUS activity depended upon the expression level of the GUS gene. The region between -377 to -290 is suggested to include the sequence required directly for high-level expression and regulation of the amyB gene.     

Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli β-glucuronidase gene and analysis of its expression in Aspergillus oryzae

Summary Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae R11340 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding -glucuronidase (GUS) downstream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.     

A repeating unit of the DYZ1 family on the human Y chromosome consists of segments with partial male-specificity

An average-sized human Y chromosome contains about 3,000 copies of the repeating DNA family DYZ1. A major repeating unit of the family, pHY10, has been cloned and an entire 3,564-bp sequence has already been determined by Nakahori et al. (1986). In the present study, pHY10 was divided into six consecutive segments, A to F, which were independently amplified by the PCR technique to see if they were strictly male-specific. pHY10 appears to consist of segments of various male-specificity. The B segment was apparently male-specific; however, the use of additional techniques (Southern-blot analysis or second PCR amplification in combination with the standard PCR) revealed homologous sequences in some females. None of the six segments of pHY10 may be male-specific in a strict sense. Different segments appear to be conserved during evolution to different extents. The 323-bp E segment appears to be the least conserved and to be responsible for the generation of most variations within the DYZ1 family.     

The superoxide-generating respiratory burst oxidase of human neutrophil plasma membrane. Phosphatidylserine as an effector of the activated enzyme

The superoxide-generating respiratory burst oxidase is an integral membrane enzyme found in the plasma membrane of polymorphonuclear leukocytes (neutrophils). NADPH-dependent superoxide generation is seen in isolated plasma membranes and in their detergent extracts following activation of the intact cells with phorbol myristate acetate. We have herein examined the effects of phospholipids on the activity of the solubilized oxidase. Solubilization of plasma membranes with 0.5% each of Tween 20 plus deoxycholate resulted in an approximately 2-fold enhancement of activity. Inclusion of phospholipids in the extraction medium resulted in further activation. At 1.0 mg/ml the order of effectiveness was phosphatidylserine (PS) greater than cardiolipin greater than phosphatidylethanolamine greater than phosphatidylinositol; phosphatidylcholine and phosphorylated inositol lipids were not effective. The concentrations required for half-maximal activation by PS and phosphatidylethanolamine were 85 and 200 micrograms/ml, respectively. When PS was used at a maximally activating concentration (0.5 mg/ml), the activity was enhanced 3-5-fold. Detergent solubilization alone elevated the Km of the oxidase for NADPH from 68 microM in intact plasma membranes to 123 microM, but inclusion of PS with detergent restored the Km to near or below that seen in intact membranes. PS also increased the Vmax by a factor of 2-3, but had no effect on the pH optimum. A plot of the activity versus enzyme concentration was linear when membranes were used, but activity showed a quadratic dependence on concentration in solubilized membrane, with lower than expected activity at lower enzyme concentration. PS restored linearity of the concentration-activity plot. The activation by PS was not influenced by the addition of Ca2+, EGTA, or dioctanoylglycerol, indicating that activation was not dependent on protein kinase C. These results implicate phosphatidylserine as a direct effector of the NADPH-oxidase.     

The adenine nucleotide binding site on yeast hexokinase PII. Affinity labeling of Lys-111 by pyridoxal 5'-diphospho-5'-adenosine

The adenine nucleotide analog, [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), is shown to be a potent and specific inhibitor of yeast hexokinase PII. Evidence that the analog binds specifically at the ATP binding site includes the demonstration that glucose binding enhances PLP-AMP binding and that PLP-AMP and ATP bind competitively with an apparent Ki(PLP-AMP) = 23 microM. In addition, from the relationship between the degree of inhibition and extent of modification, it is estimated that the incorporation of 1 mol of PLP-AMP/mol of subunit is required for complete inhibition. Borohydride reduction of the Schiff's base complex formed between hexokinase and [3H]PLP-AMP gives a stable product. The reduced derivative was digested with trypsin and a single radioactive peptide was isolated by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis identified Lys-111 as the modified residue. Taking into account the known structures of the binary complexes (Shoham, M., and Steitz, T. A. (1980) J. Mol. Biol. 140, 1-14), the results suggest that Lys-111, located in the smaller of the two lobes of hexokinase, moves into the active site upon formation of the ternary complex.     

Variation in karyotypes of Dugesia japonica japonica (Turbellaria) from Ôsaka Prefecture,Central Japan

An analysis of the karyotypes of Dugesia japonica japonica Ichikawa et Kawakatsu, 1964, from 30 localities of seven river systems in Ôsaka Prefecture, Central Japan, revealed a total of 26 karyotypes of which 12 are new-found varieties. More than two karyotypes were found in many localities. The mixoaneuploidic triploid karyotype showed the widest distribution (21 localities), orthoploidic diploid karyotype and orthoploidic mixoploid of diploid and triploid karyotype were next (13 localities, respectively), and other karyotypes (triploidic aneuploid, orthoploidic triploid and mixoploid of triploid & tetraploid) were also found. Some correlation of karyotype with elevation was also detected.