The effect of clomiphene citrate on ovulation and spawning in indomethacin treated carp, Cyprinus carpio

Studies were conducted on the effect of clomiphene on ovulation and spawning in mature C. carpio pre-treated with indomethacin. It was demonstrated that indomethacin caused a block to ovulation and spawning at two dose levels (5 and 10 μg⁻¹g). This blockade could be overcome by two (10 μg⁻¹g each) successive dosages of clomiphene given 7 and 31 h after the indomethacin treatment. When indomethacin and clomiphene treatments were given concomittantly, the inhibitory effects of indomethacin remained more pronounced.     

Phosphatases revisited: analysis of particle-associated enzyme activities in aquatic systems

A modified assay for alkaline or acid phosphatases associated with microorganisms in aquatic environments has been developed. This is based on collecting microplankton on filters and subsequently determining the enzyme activities spectrophotometrically or fluorometrically, using p-nitrophenyl-phosphate or 4-methylumbelliferyl phosphate, respectively as substrates. The assay is simple and rapid, and has the further advantage of permitting phosphatase activities to be assigned to specific size fractions of the natural microplankton. In samples taken from Lake Kinneret and a nearby reservoir, a consistently high proportion of the total alkaline or acid phosphatase activity was associated with the size fraction < 0.8 µm > 0.2 µm indicating the potentially high contribution of bacteria to these activities. This approach can also be used to examine the enzymatic potential of microplankton to release orthophosphate from other organo-phosphate substrates.     

Strategy for statistical-mapping of potential regulatory regions in the human genome

We have explored the feasibility of using crude nuclear extracts and band-shift experiments, to select and clone short human DNA fragments that contain binding sites for sequence-specific nuclear proteins. We show that this strategy is feasible. It provides a means to systematically identify the recognition sequences of proteins found in nuclear extracts prepared from various tissues, in order to compile a protein-binding-sequence catalogue. A complete catalogue would provide the data needed for searching the "raw" human DNA sequences for occurrence of protein-binding sites and thus, to construct a statistical map of potential regulatory regions in the human genome.     

Proteomics of glycoproteins based on affinity selection of glycopeptides from tryptic digests

Identification of glycoproteins in complex mixtures derived from either human blood serum or a cancer cell line was achieved in a process involving the steps of (1) reduction and alkylation, (2) proteolysis of all proteins in the mixture with trypsin, (3) affinity chromatographic selection of the glycopeptides with an immobilized lectin, (4) direct transfer of the glycopeptide fraction to a reversed-phase liquid chromatography (RPLC) column and further fractionation by gradient elution, (5) matrix-assisted laser desorption ionization mass spectrometry of individual fractions collected from the RPLC column, and (6) peptide identification based on a database search. The types of glycoproteins analyzed were; (1) N-type glycoproteins of known primary structure, (2) N-type glycoproteins of unknown structure, and (3) O-type glycoproteins glycosylated with a single N-acetylglucosamine. Identification of peptides from complex mixtures was greatly facilitated by either C-terminal sequencing with a carboxypeptidase mixture or by comparing chromatographic behavior and mass to standards, as in the case of a known protein. In addition, deglycosylation of peptides with N glycosidase F was necessary to identify N-type glycoproteins of unknown structure. The strength of this approach is that it is fast and targets specific molecular species or classes of glycoproteins for identification. The weakness is that it does not discriminate between glycoforms.     

The GANC tetraloop: a novel motif in the group IIC intron structure

Tetraloops are a common building block for RNA tertiary structure, and most tetraloops fall into one of three well-characterized classes: GNRA, UNCG, and CUYG. Here, we present the sequence and structure of a fourth highly conserved class of tetraloop that occurs only within the ζ-ζ′ interaction of group IIC introns. This GANC tetraloop was identified, along with an unusual cognate receptor, in the crystal structure of the group IIC intron and through phylogenetic analysis of intron RNA sequence alignments. Unlike conventional tetraloop-receptor interactions, which are stabilized by extensive hydrogen-bonding interactions, the GANC-receptor interaction is limited to a single base stack between the conserved adenosine of the tetraloop and a single purine of the receptor, which consists of a one- to three-nucleotide bulge and does not contain an A-platform. Unlike GNRA tetraloops, the GANC tetraloop forms a sharp angle relative to the adjacent helix, bending by approximately 45° toward the major groove side of the helix. These structural attributes allow GANC tetraloops to fit precisely within the group IIC intron core, thereby demonstrating that structural motifs can adapt to function in a specific niche.     

Congenic mapping of a blood pressure QTL region on rat chromosome 10 using the Dahl salt-sensitive rat with introgressed alleles from the Milan normotensive strain

Multiple blood pressure (BP) quantitative trait loci (QTLs) are reported on rat chromosome 10 (RNO10). Of these, QTLs detected by contrasting the genome of the hypertensive Dahl salt-sensitive (S) rat with two different relatively normotensive strains, Lewis (LEW) and the Milan normotensive strain (MNS), are reported. Because the deduced QTL regions of both S vs. LEW and S vs. MNS comparisons are within large genomic segments encompassing more than 2 cM, there was a need to further localize these QTLs and determine whether the QTLs are unique to specific strain comparisons. Previously, the S.MNS QTL1 was mapped to less than 2.6 cM as a differential segment between two congenic strains. In this study, multiple congenic strains spanning the projected interval were studied. The BP effect of each strain was interpreted as the net effect of alleles introgressed within that congenic strain. The results suggest that the MNS alleles within the previously proposed differential segment (D10Rat27-D10Rat24) do not independently lower BP of the S rat. However, another congenic strain, S.MNS(10) × 9, containing introgressed MNS alleles that are outside of the previously proposed differential segment is of interest because (1) it demonstrated a BP-lowering effect, (2) it is contained within a single congenic strain and is not based on the observed effect of a differential segment, and, more importantly, (3) it overlaps with the previously identified S.LEW BP QTL region. Identification of the same QTL affecting BP in multiple rat strains will provide further support for the QTL’s involvement and importance in human essential hypertension.     

Anti-arrhythmic effects of cyclopiazonic acid in Langendorff-perfused murine hearts

We investigated the effects of reducing sarcoplasmic reticular (SR) Ca²⁺ stores using the Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA) in Langendorff-perfused mouse hearts exposed to different pro-arrhythmic agents all known to produce Ca²⁺-mediated arrhythmogenesis. CPA (100 and 150 nM) produced progressive (beginning over 1 min) and significant (P < 0.0001) reductions in peak amplitudes of Ca²⁺ transients evoked by regular stimulation in isolated Fluo-3 loaded myocytes from F/F₀ = 3.2 ± 0.16 (n = 12 cells) to 1.62 ± 0.012 (n = 6 cells) and 1.53 ± 0.06 (n = 12 cells), respectively, consistent with previous reports describing reductions of store Ca²⁺ in other cell systems. The corresponding effects of CPA were then examined in intact hearts exposed to isoproterenol (100 nM), elevated extracellular [Ca²⁺] (5 mM) and caffeine (1 mM). All three agents produced ventricular tachycardia either when added alone or simultaneously with CPA during programmed electrical stimulation. However, arrhythmogenicity was not observed when such agents were added 10 min after introduction of CPA. CPA thus antagonized this Ca²⁺-mediated arrhythmogenesis but only under circumstances of SR Ca²⁺ depletion. These alterations in arrhythmogenic tendency took place despite an absence of alterations in electrogram and monophasic action potential characteristics. This was in sharp contrast to previous observations in murine, ΔKPQ-Scn5a (LQT3) and KCNE1^−/− (LQT5), systems where re-entry has been implicated in arrhythmogenesis.     

Room temperature photooxidation of β-carotene and peripheral chlorophyll in photosystem II reaction centre

Differential kinetic absorption spectra were measured during actinic illumination of photosystem II reaction centres and core complexes in the presence of electron acceptors silicomolybdate and ferricyanide. The spectra of samples with ferricyanide differ from those with both ferricyanide and silicomolybdate. Near-infrared spectra show temporary beta-carotene and peripheral chlorophyll oxidation during room temperature actinic illumination. Peripheral chlorophyll is photooxidized even after decay of beta-carotene oxidation activity and significant reduction of beta-carotene content in both reaction centres and photosystem II core complexes. Besides, new carotenoid cation is observed after about 1 s of actinic illumination in the reaction centres when silicomolybdate is present. Similar result was observed in PSII core complexes. HPLC analyses of illuminated reaction centres reveal several novel carotenoids, whereas no new carotenoid species were observed in HPLC of illuminated core complexes. Our data support the proposal that pigments of inner antenna are a sink of cations originating in the photosystem II reaction centre.