Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Antisense inhibition of the Nr gene restores normal ripening to the tomato Never-ripe mutant, consistent with the ethylene receptor-inhibition model

The hormone ethylene regulates many aspects of plant growth and development, including fruit ripening. In transgenic tomato (Lycopersicon esculentum) plants, antisense inhibition of ethylene biosynthetic genes results in inhibited or delayed ripening. The dominant tomato mutant, Never-ripe (Nr), is insensitive to ethylene and fruit fail to ripen. The Nr phenotype results from mutation of the ethylene receptor encoded by the NR gene, such that it can no longer bind the hormone. NR has homology to the Arabidopsis ethylene receptors. Studies on ethylene perception in Arabidopsis have demonstrated that receptors operate by a "receptor inhibition" mode of action, in which they actively repress ethylene responses in the absence of the hormone, and are inactive when bound to ethylene. In ripening tomato fruit, expression of NR is highly regulated, increasing in expression at the onset of ripening, coincident with increased ethylene production. This expression suggests a requirement for the NR gene product during the ripening process, and implies that ethylene signaling via the tomato NR receptor might not operate by receptor inhibition. We used antisense inhibition to investigate the role of NR in ripening tomato fruit and determine its mode of action. We demonstrate restoration of normal ripening in Nr fruit by inhibition of the mutant Nr gene, indicating that this receptor is not required for normal ripening, and confirming receptor inhibition as the mode of action of the NR protein.     

Kinetics of incorporation of O6-methyldeoxyguanosine monophosphate during in vitro DNA synthesis

O6-Methyldeoxyguanosine triphosphate (m6dGTP), known to be produced in vivo by methylation of deoxyguanosine triphosphate with simple methylating mutagens, is utilized by prokaryotic DNA polymerases during in vitro replication of synthetic and natural DNA template-primers. A study of the kinetic behavior of m6dGTP during DNA replication in vitro and of its effect on DNA replication indicates that m6dGTP acts as an analogue of dATP with Kappm of about 6 microM for Escherichia coli DNA polymerase I (Klenow fragment) compared to the Kappm of about 0.8 microM for dATP. m6dGTP is not incorporated in the complete absence of dATP (a competitive inhibitor). m6dGTP also inhibits in vitro DNA synthesis. Different DNA polymerases behave differently in utilization and turnover of m6dGTP. T4 DNA polymerase shows stronger discrimination against m6dGMP incorporation than either T5 DNA polymerase or E. coli DNA polymerase I. The possibility that m6dGTP is unlikely to contribute significantly to in vivo mutation is discussed.     

In vitro capacitation and fertilizing ability of ejaculated rabbit sperm treated with lysophosphatidylcholine

Four experiments were replicated 1) to establish dose-response relationships between lysophosphatidylcholine (LPC), sperm motility, and the acrosome reaction (AR), 2) to evaluate the influence of rabbit serum (RS) on these endpoints, 3) to compare buck differences in induction of the AR, and 4) to examine fertilizing ability in vitro of sperm tested under the first three objectives. Semen was collected from Dutch-belted rabbits, washed once by centrifugation, resuspended, and preincubated for 2 or 4 hr in a chemically defined medium (DM), DM plus 20% RS, or BSA-free DM plus 20% RS at 37°C. At the end of preincubation LPC was added to the preincubated sperm at concentrations of from 0 to 100 μg/ml. Sperm were examined .5–4 hr later for AR and sperm motility. For in vitro fertilization, sperm and ova were coincubated in DM up to 24 hr after insemination and in a more complex medium for another 24 hr. Addition of LPC to 4-hr-preincubated sperm was more effective for inducing the AR than addition to 2-hr-preincubated sperm. A significant increase (P < .05) in the AR occurred in 15 and 30 min following exposure to 100 and 80 μg of LPC per ml, respectively, but the higher concentration of LPC decreased sperm motility. Addition of 20% RS to DM with or without BSA surprisingly inhibited the AR but maintained sperm motility, as expected. Bucks differed (P < .05) in the initial percentage and the induced percentage of AR sperm. For the AR the optimal concentration of LPC per ml was 80 μg, but for in vitro fertilization 60 μg tended to be superior.     

“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.     

Early singers attend to conspecific but not heterospecific behavioural cues at dawn

During the dawn chorus songbirds initiate singing activities just prior to sunrise. Environmental factors affect the timing of the dawn chorus, but relatively little is known about how behavioural cues influence chorus timing. We assessed whether early playback of conspecific and heterospecific song influenced chorus start times in two early‐singing temperate species, hermit thrush Catharus guttatus and veery C. fuscescens. We used arrays of GPS time‐synchronized recorders to record the dawn chorus at 30 locations. In each location, thirty minutes prior to natural chorus initiation, we broadcast: 1) an early singing species (hermit thrush or veery), 2) a late singing species (black‐throated green warbler Setophaga virens or Nashville warbler Oreothlypis ruficapilla), 3) a noise control, and 4) also recorded without playback (silent control). Playback treatments were separated by two‐three days at each location. Both hermit thrush and veery sang significantly earlier in response to conspecific playback compared to noise or silent controls. Both species also sang earlier in response to conspecific playback compared to the day before or the day after playback. Neither species sang earlier in response to heterospecific playback (regardless of whether the broadcast species was a naturally early or late song initiator) compared to noise or silent controls. Our results support the theory that the dawn chorus is a communication network where individuals attend primarily to conspecific cues; heterospecific song appears to have minimal impacts on the chorus start times of the two early‐singing species we investigated.     

On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.     

Site-directed mutagenesis to determine essential residues of ribulose-bisphosphate carboxylase ofRhodospirillum rubrum

Both Lys-166 and His-291 of ribulosebisphosphate carboxylase/oxygenase fromRhodospirillum rubrum have been implicated as the active-site residue that initiates catalysis. To decide between these two candidates, we resorted to site-directed mutagenesis to replace Lys-166 and His-291 with several amino acids. All 7 of the position-166 mutants tested are severely deficient in carboxylase activity, whereas the alanine and serine mutants at position 291 are ∼40% and ∼18% as active as the native carboxylase, essentially ruling out His-291 in theRhodospirillum rubrum carboxylase (and by inference His-298 in the spinach enzyme) as a catalytically essential residue. The ability of some of the mutant proteins to undergo carbamate formation or to bind either ribulosebisphosphate or a transition-state analogue remains largely unimpaired. This implies that Lys-166 is not required for substrate binding; rather, the results corroborate the earlier postulate that Lys-166 functions as an acid-base group in catalysis or in stabilizing a transition state in the reaction pathway.     

Development of reproductive biotechnologies in domestic animals from artificial insemination to cloning: a perspective

There has been a remarkable increase in knowledge resulting in the application of reproductive biotechnologies in animals, with profound implications for human beings. The major accomplishments in domestic animals, particularly with cattle, are reviewed here. An attempt is made to examine these, in perspective, to indicate the steps by which progress was made, and to appreciate that the explosion of new findings today would not have been possible without the careful studies of yesteryears.     

Induction and characterization of Ph1 wheat mutants

The cloning of genes for complex traits in polyploid plants that possess large genomes, such as hexaploid wheat, requires an efficient strategy. We present here one such strategy focusing on the homologous pairing suppressor (Ph1) locus of wheat. This locus has been shown to affect both premeiotic and meiotic processes, possibly suggesting a complex control. The strategy combined the identification of lines carrying specific deletions using multiplex PCR screening of fast-neutron irradiated wheat populations with the approach of physically mapping the region in the rice genome equivalent to the deletion to reveal its gene content. As a result, we have located the Ph1 factor controlling the euploid-like level of homologous chromosome pairing to the region between two loci (Xrgc846 and Xpsr150A). These loci are located within 400 kb of each other in the rice genome. By sequencing this region of the rice genome, it should now be possible to define the nature of this factor.