Murine progesterone receptor exists predominantly as the 83-kilodalton 'A' form

Progesterone receptors (PgR) are known to exist in two molecular forms commonly designated as 'A' and 'B' forms, and the relative ratio of these two forms has been shown to vary among species. Although the rodent systems were some of the earliest experimental systems used to examine the regulation of PgR, as yet very little is known concerning the molecular composition of PgR in this species. Accordingly, to define the relative ratio of 'A' and 'B' forms in murine PgR, we have analyzed tissue extracts from normal, ovariectomized, and estradiol treated animals by photoaffinity labeling and immunoblotting techniques using a variety of anti-PgR antibodies. Under all experimental conditions, two forms of PgR with approximate molecular weights of 115 kDa ('B' form) and 83 kDa ('A' form) were found. In all tissues examined, the 83 kDa 'A' form was predominant, and this was independent of the hormonal status of the animal and different buffers used to prepare tissue extracts. In uterus the ratio of 'A' to 'B' was 3:1, in vagina it was 2:1, and in mammary glands it more closely resembled the uterus. This leads us to conclude that murine PgR exists predominantly as the 83-kDa 'A' form which may represent a general characteristic of rodent PgR. In this species there may also be some tissue specificity with regard to the absolute ratio of the two forms of PgR.     

Solvent interactions stabilising nucleic acid conformers

The transition of oligonucleotides from the B to the A conformation has been studied by the use of simple geometric calculations aimed at finding possible hydration sites which could stabilize these conformations. The method involves the classification of equally spaced grid points, surrounding the oligonucleotide, into groups depending on whether a water molecule, so placed, could form single, multiple or nil contacts to polar oligonucleotide atoms. The occurrence of the multiple, and therefore bridging, sites is more extensive for the 'A' than the 'B' conformation. Thus, more general evidence is presented in support of the economy of hydration hypothesis in which phosphate groups, in the low humidity 'A' form, have been seen to be bridged by single water molecules. Similar calculations for the 'Z' DNA conformation show a different preference for multiple bridging sites.     

The stereospecificity of the reduction of nitrate by reduced nicotinamide-adenine dinucleotides catalysed by Candida utilis preparations.

The reduction of nitrate by reduced nicotinamide-adenine dinucleotides, catalysed by extract of Candida utilis, exhibits an apparent high degree of stereospecificity for the 'B' methylene hydrogen atom of NADPH and mixed stereospecificity for the methylene hydrogen atoms of NADH. Purified nitrate reductase, on the other hand, exhibits 'A' stereospecificity for NADH and NADPH. The apparent switch of stereospecificity from the 'B' to the 'A' side of NADPH, which occurs after purification of the enzyme, is partly explained by the fact that in crude extracts nitrate is reduced completely to ammonia. Nitrite does not accumulate but is reduced to ammonia by nitrite dehydrogenase, which is 'B'-specific, so that up to 75% of hydrogen removed from NADPH during the reduction of nitrate could occur from the 'B' side. A further increase in the removal of hydrogen from the 'B' side of NADPH could be the kinetic isotope effect that is observed when ['A'-3H]NADPH is the reductant, the H--C bond being cleaved 2.3 times faster than the 3H--C bond. The mixed stereospecificity observed with NADH has been traced to an uncharacterized enzyme that catalyses a 'B'-specific exchange between NAD+ and NADH. This reaction is discussed in relation to the possibility that it may explain other cases of apparent mixed stereospecificity that have been reported.     

''A'' forms of RNAs in single strands, duplexes and RNA-DNA hybrids.

Helical parameters have been calculated for the 'A' form minimum energy conformations of ApA, CpC, GpG, UpU, GpC and UpA. The helix geometries are base sequence dependent. The single strands are narrower and more tightly wound than that duplex RNA-11 form. 9-12 kcal./mole are needed to convert these single strands to the RNA-11 conformation. However, in some sequences other 'A' type conformers capable of complementary base pairing may be formed at lower energetic cost. There is substantially more base stacking in the calculated single strands than in the RNA-11 conformation. Calculated intrastrand base stacking energies reflect these differences, and also are sequence dependent. The 'A' form RNA subunits differ from the analogous DNAs in possessing a larger rise per residue, needed to accomodate the 2'-OH. RNA-DNA hybrids are consequently more likely to be in the 'A-RNA than in the 'A'-DNA conformation, although the base sequence determines the extent of the preference.     

Variations in oil palm (Elaeis guineensis Jacq.) progeny response to high aluminium concentrations in solution culture

Aluminium (Al) phytotoxicity is an important soil constraint that limits crop yield. The objectives of this study were to investigate how growth, physiology, nutrient content and organic acid concentration is affected by Al, and to assess the degree of Al tolerance in different oil palm progeny (OPP). Four OPPs ['A' (Angola dura × Angola dura), 'B' (Nigerian dura × Nigerian dura), 'C' (Deli dura × AVROS pisifera) and 'D' (Deli dura × Dumpy AVROS pisifera)] were grown in different Al concentrations (0, 100 and 200 μm) in aerated Hoagland solution, pH 4.4, for 80 days. We observed a severe reduction (57.5%) in shoot dry weight, and root tips were reduced by 46.5% in 200 μm Al. In 'B' and 'C', the majority of macro- and micronutrients in plants were reduced significantly by 200 μm Al, with Mg being lowered by more than 50% in roots and shoots. The 200 μm Al treatment resulted in a 56.50% reduction in total leaf area, a 20% reduction in net photosynthesis and a 17% reduction in SPAD chlorophyll value in the third leaf. Root tips (0-5 mm) showed a significant increase in oxalic acid content with increasing Al concentration (～ 5.86-fold); progeny 'A' had the highest concentration of oxalic acid. There was a significant interaction between Al concentration × OPP on total leaf number, root volume, lateral root length, Mg and K in root and shoot tissues, and Ca and N in shoots. The OPPs could be ranked in their tolerance to Al as: 'A' > 'D' > 'B' > 'C'.     

Studies of the action of symmetrical and mixed disulphide thiol-modifying reagents on the esterase activity of cytoplasmic aldehyde dehydrogenase

The effect of various thiol-modifying reagents on the esterase activity of sheep liver cytoplasmic aldehyde dehydrogenase is reported here. Both symmetrical reagents (disulfiram, 2,2'- and 4,4'-dithiodipyridines) and unsymmetrical reagents (methyl diethylthiocarbamyl disulphide, methyl 2- and 4-pyridyl disulphides) were investigated. The results suggest that all the modifiers react to varying extents with a pair of enzymic thiol groups ('A' and 'B'), and that the more specifically group 'A' is modified, the more the enzyme is inactivated. This supports the idea that group 'A' may be the essential nucleophile in the reaction catalysed by aldehyde dehydrogenase. Modification of group 'B' may or may not reduce the esterase activity depending on the nature of the label introduced. The results of the present experiments and of previous similar experiments concerning the dehydrogenase activity of the enzyme are consistent with the proposal that a common active site is responsible for both esterase and dehydrogenase activities.     

A model for the single stranded random coil form of polydeoxyadenylic acid from minimum energy conformations of the dimeric subunit.

The minimum energy conformations of dApdA have been examined for their suitability as buildings blocks of the single stranded coil form of polynucleotides. Calculations of the characteristic ratio C difference = less than ro greater than 2/n liter2 were made for a polymer generated from all the low energy conformers, as well as for selected combinations. A polymer composed of a conformer with omega', omega = t*,g+,(skewed) psi = t, C-(2)-endo type pucker, in combination with the 'B' form, has a C difference equal to that observed in coils of apurinic acid (6) when the fraction of 'B' form conformers is approximately 25% and approximately 91%. The t*,g+ conformer is the second lowest energy form in the C-(2)-endo puckering domain, following the 'B' form.     

The structure and cytochemistry of the neurosecretory cells in the intertidal gastropod Thais bufo (lamarck)

Three types of neurosecretory cells ('A', 'B' and 'C' cells) have been distinguished in the central ganglion of Thais bufo. A few homogenous groups are met with and the rest are all heterogenous groups. The histochemical observations reveal that the neurosecretory material is rich in carbohydrates, disulphides, protein bound amino groups, glycoprotein and lipids. Thus the neurosecretory material seems to be a lipoprotein--glycoprotein complex.     

Quantitative structure-activity relationship of phenoxyphenyl-methanamine compounds with 5HT2A, SERT, and hERG activities

QSAR models have been used to evaluate activities for compounds in the phenoxyphenyl-methanamine (PPMA) class of compounds. These models utilize Hammett-type donating-withdrawing substituent values as well as simple parameters to describe substituent size and elucidate the SAR of the 'A' and 'B' rings. Using this methodology, intuitive QSAR relationships were found for the three biological activities with R(2) values of 0.73, 0.45, and 0.58 for 5HT(2A), SerT, and hERG activities.     

Cloning of chromosomal beta-lactamase genes from Yersinia enterocolitica

Two beta-lactamase genes present in the chromosome of Yersinia enterocolitica have been cloned individually into the plasmid pACY184 and expressed in Escherichia coli. The gene for broad-spectrum beta-lactamase I ('A') was cloned from a strain belonging to the O:3 serotype, and the gene for (cephalosporinase) beta-lactamase II ('B') was cloned from a strain of the O:5b serotype. The properties of the beta-lactamases expressed in E. coli are similar to those previously described in Y. enterocolitica.     

A new superfamily of putative NTP-binding domains encoded by genomes of small DNA and RNA viruses

Statistically significant similarity was revealed between amino acid sequences of NTP-binding pattern-containing domains which are among the most conserved protein segments in dissimilar groups of ss and dsDNA viruses (papova-, parvo-, geminiviruses and P4 bacteriophage), and RNA viruses (picorna-, como- and nepoviruses) with small genomes. Within the aligned domains of 100-120 amino acid residues, three highly conserved sequence segments have been identified, i.e. 'A' and 'B' motifs of the NTP-binding pattern, and a third, C-terminal motif 'C', not described previously. The sequence of the 'B' motif in the proteins of the new superfamily is unusually variable, with substitutions, in some of the members, of the Asp residue conserved in other NTP-binding proteins. The 'C' motif is characterized by an invariant Asn residue preceded by a stretch of hydrophobic residues. As the new superfamily included a well studied DNA and RNA helicase, T antigen of SV40, helicase function could be tentatively assigned also to the other related viral putative NTP-binding proteins. On the other hand, the possibility of different and/or multiple functions for some of these proteins is discussed.     

Neural cell fate in rca1 and cycA mutants: the roles of intrinsic and extrinsic factors in asymmetric division in the Drosophila central nervous system.

In the central nervous system (CNS) of Drosophila embryos lacking regulator of cyclin A (rca1) or cyclin A, we observe that several ganglion mother cells (GMCs) fail to divide. Whereas GMCs normally produce two sibling neurons that acquire different fates ('A/B'), non-dividing GMCs differentiate exclusively in the manner of one of their progeny ('B'). In zygotic numb mutants, sibling neuron fate alterations ('A/B' to 'A/A') occur infrequently or do not occur in some sibling pairs; we have determined that depletion of both maternal and zygotic numb causes sibling neurons to acquire equalized fates ('A/A') with near-complete expressivity. In rca1, numb mutant embryos, we observe binary cell fate changes ('B' to 'A') in several GMCs as well. Finally, we have demonstrated that expression of Delta in the mesoderm is sufficient to attain both sibling fates. Our results indicate that the intrinsic determinant Numb is absolutely required to attain differential sibling neuron fates. While the extrinsic factors Notch and Delta are also required to attain both fates, our results indicate that Delta signal can be received from outside the sibling pair.     

DNA double helix destabilizing properties of cyclobisintercaland compounds and competition with a single strand binding protein

The DNA helix destabilizing activity of a series of cyclobisintercaland compounds (CBIs) has been evaluated by measuring their ability to displace a 32P-labelled oligonucleotide primer (17-mer) hybridized to the single stranded DNA of M13. This destabilizing activity appears to be strongly dependent on the cyclic structure (the linear acyclic references are inactive) and the size of the macrocycle; both features being known to determine the preferential binding of the compound to ssDNA. Interestingly, CBIs induced the dissociation of the duplex template in a concentration range (0.5-1 microM) close to that required for the destabilizing activity of single stranded DNA binding proteins (SSBs). Therefore competition experiments between CBIs and an SSB protein (Eco SSB) for binding to a single stranded oligonucleotide target (36-mer) have been performed through gel electrophoresis and nitrocellulose binding assays and strong inhibitory effects on the formation of the SSB:36-mer complex have been observed.     

An attempt to improve the flavour of 'nono' by the use of starter cultres

Three close-fitting lid fermenters were incubated at 27 ± 2°C for 120 h. 'A' served as a control and contained 100 ml raw goats' milk; 'B' contained 50 ml 48-h-old nono as starter culture plus 50 ml pasteurized milk; and 'C' contained 100 ml pasteurized milk only. The pH and lactic acid contents were determined at intervals. Fermenter 'B' gave the best results in that the milk reached the lowest pH values and the highest lactic acid contents, and this resulted in a more appetizing and bitter product.     

Computed free energy differences between point mutations in a collagen-like peptide

We studied the results of mutating alanine --> glycine at three positions of a collagen-like peptide in an effort to develop a computational method for predicting the energetic and structural effects of a single point genetic mutation in collagen, which is associated with the clinical diagnosis of Osteogenesis Imperfecta (OI). The differences in free energy of denaturation were calculated between the collagen-like peptides [(POG)(4)(POA)(POG)(4)](3) and [(POG)(10)](3) (POG: proline-hydroxyproline-glycine).* Our computational results, which suggest significant destabilization of the collagen-like triple-helix upon the glycine --> alanine mutations, correlate very well with the experimental free energies of denaturation. The robustness of our collagen-like peptide model is shown by its reproduction of experimental results with both different simulation paths and different lengths of the model peptide. The individual free energy for each alanine --> glycine mutation (and the reverse free energy, glycine --> alanine mutation) in the collagen-like peptide has been calculated. We find that the first alanine introduced into the triple helix causes a very large destabilization of the helix, but the last alanine introduced into the same position of an adjacent chain causes a very small change in the peptide stability. Thus, our results demonstrate that each mutation does not contribute equally to the free energy. We find that the sum of the calculated individual residues' free energy can accurately model the experimental free energy for the whole peptide.     

UDP-glucose dehydrogenase: substrate binding stoichiometry and affinity

Precise structural parameters of polyribonucleotides single stranded helices are determined as well as those of double stranded helices of poly 2′-O-methyl A and of poly A at neutral and acid pH. Infrared linear dichroism investigations indicate the similarity of the conformation of the sugar-phosphate backbone of these single and double stranded helices. The angles of the phosphate group for single stranded helix at neutral pH is found to be oriented at 48° for the 02P02 bisector and at about 65° for the 02–03 line to the helix axis. Similar values were found for double stranded poly A helix at acid pH. These structural parameters obtained for the first time on single stranded polynucleotide helices are proposed to be valid for other similar helical chains such as poly A segments of nuclear or messenger RNA and single stranded CCA acceptor end of transfer RNA.     

Consensus sequences for good and poor removal of uracil from double stranded DNA by uracil-DNA glycosylase.

We have purified uracil DNA-glycosylase (UDG) from calf thymus 32,000-fold and studied its biochemical properties, including sequence specificity. The enzyme is apparently closely related to human UDG, since it was recognised by a polyclonal antibody directed towards human UDG. SDS-PAGE and western analysis indicate an apparent M(r) = 27,500. Bovine UDG has a 1.7-fold preference for single stranded over double stranded DNA as a substrate. Sequence specificity for uracil removal from dsDNA was examined for bovine and Escherichia coli UDG, using DNA containing less than one dUMP residue per 100 nucleotides and synthetic oligonucleotides containing one dUMP residue. Comparative studies involving about 40 uracil sites indicated similar specificities for both UDGs. We found more than a 10-fold difference in rates of uracil removal between different sequences. 5'-G/CUT-3' and 5'-G/CUG/C-3' were consensus sequences for poor repair whereas 5'-A/TUAA/T-3' was a consensus for good repair. Sequence specificity was verified in double stranded oligonucleotides, but not in single stranded ones, suggesting that the structure of the double stranded DNA helix has influence on sequence specificity. Rate of uracil removal appeared to be slightly faster from U:A base pairs as compared to U:G mis-matches. The results indicate that sequence specific repair may be a determinant to be considered in mutagenesis.     

A high-throughput method for the detection of homoeologous gene deletions in hexaploid wheat

Background  

Mutational inactivation of plant genes is an essential tool in gene function studies. Plants with inactivated or deleted genes may also be exploited for crop improvement if such mutations/deletions produce a desirable agronomical and/or quality phenotype. However, the use of mutational gene inactivation/deletion has been impeded in polyploid plant species by genetic redundancy, as polyploids contain multiple copies of the same genes (homoeologous genes) encoded by each of the ancestral genomes. Similar to many other crop plants, bread wheat (Triticum aestivum L.) is polyploid; specifically allohexaploid possessing three progenitor genomes designated as 'A', 'B', and 'D'. Recently modified TILLING protocols have been developed specifically for mutation detection in wheat. Whilst extremely powerful in detecting single nucleotide changes and small deletions, these methods are not suitable for detecting whole gene deletions. Therefore, high-throughput methods for screening of candidate homoeologous gene deletions are needed for application to wheat populations generated by the use of certain mutagenic agents (e.g. heavy ion irradiation) that frequently generate whole-gene deletions.