Hypomethylated sequences: Characterization of the duplicate soybean genome

Soybean is believed to be a diploidized tetraploid generated from an allotetraploid ancestor. In this study, we used hypomethylated genomic DNA as a source of probes to investigate the genomic structure and methylation patterns of duplicated sequences. Forty-five genomic clones from Phaseolus vulgaris and 664 genomic clones from Glycine max were used to examine the duplicated regions in the soybean genome. Southern analysis of genomic DNA using probes from both sources revealed that greater than 15% of the hypomethylated genomic regions were only present once in the soybean genome. The remaining ca. 85% of the hypomethylated regions comprise duplicated or middle repetitive DNA sequences. If only the ratio of single to duplicate probe patterns is considered, it appears that 25% of the single-copy sequences have been lost. By using a subset of probes that only detected duplicated sequences, we examined the methylation status of the homeologous genomes with the restriction enzymes MspI and HpaII. We found that in all cases both copies of these regions were hypomethylated, although there were examples of low-level methylation. It appears that duplicate sequences are being eliminated in the diploidization process. Our data reveal no evidence that duplicated sequences are being silenced by inactivation correlated with methylation patterns.     

Forskolin and 3-Isobutyl-l-Methylxanthine Increase Basal and Sodium Nitroprusside-Elevated Cyclic GMP Levels in Adult Guinea-Pig Cerebellar Slices

Abstract: In this study, the interaction between 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) in [³H]adenine-or [³H]-guanine-prelabelled adult guinea-pig cerebellar slices was investigated. Basal levels of [³H]cGMP were enhanced by forskolin, although no plateau was reached over the concentration range tested (0.1-100 μM). However, forskolin elicited a concentration-dependent, saturable potentiation of sodium nitroprusside (SNP)-stimulated [³H]cGMP accumulation (forskolin EC₅₀ value of 0.98 β 0.23 μM; 10 μM forskolin produced a 1.8 β 0.3-fold potentiation of the SNP response at 2.5 min). The forskolin potentiation was observed at all concentrations of SNP tested (0.001-10 mM). forskolin also elicited a large stimulation of [³H]-cAMP in [³H]adenine-prelabelled guinea-pig cerebellar slices; however, 1,9-dideoxyforskolin failed to elicit either a [³H]cAMP response or a potentiation of the SNP-induced [³H]cGMP response at concentrations up to 100 μM. Pretreatment with oxyhaemoglobin (50 μM) inhibited the response to SNP (1 mM) and forskolin (10 μM), as well as the response evoked by the combination of SNP and forskolih. A^G-Nitro-l -arginine (100 μM) inhibited the response to forskolin alone, but did not change the response to SNP or the potentiation induced by forskolin on SNP-induced [³H]cGMP levels. The protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7; 100 μM), staurosporine (10 μM), polymyxin B (100 μM), and Ro 31-8220 (10 μM) had no effect on the [³H]cGMP response to either SNP or the combination of SNP plus forskolin. N⁶,2′-Dibutyryl cAMP, at concentrations up to 10 mM, was also without effect on [³H]cGMP levels induced by SNP. 3-lso-butyl-1-methylxanthine reproduced the effect of forskolin on SNP-induced [³H]cGMP levels, but a less-than-additive effect was observed when the response to SNP was studied in the presence of forskolin and 3-isobutyl-1-methylxanthine. Taken together, these results infer that crosstalk between cyclic nucleotides takes place in guinea-pig cerebellar slices, and that cAMP may regulate cGMP-mediated responses in this tissue.     

The role of infectious agents in sudden infant death syndrome

Abstract Epidemiological factors associated with susceptibility to respiratory infections are similar to those associated with Sudden Infant Death Syndrome. Here we review the evidence that respiratory pathogens might be involved in some cases of Sudden Infact Death Syndrome in the context of factors identified in epidemiological studies of cot deaths: the age range affected; mother's smoking; respiratory viral infections; immunisation status. Both laboratory and epidimiological evidence suggests that vulnerability of infants to infectious agents depends on interactions between genetic, developmental and environmental factors that contribute to colonisation by microorganisms, the inflammatory and specific immune responses and the infants' physiological responses to inflammatory mediators. A model is proposed to explain how microorganisms might trigger a series of events resulting in some of these unexpected deaths and discusses how the present recommendations regarding child care practices might help reduce the numbers of Sudden Infant Death Syndrome cases associated with infectious agents.     

Cdk inhibitors: on the threshold of checkpoints and development

The regulation of cyclin-dependent kinases is at the heart of cell cycle control and, by inference, the control of cell proliferation. Recent advances in regulation of these kinases have uncovered a group of small proteins that bind to and inhibit them, thus preventing cell cycle progression. Linking these proteins to tumor suppressor functions has provided a much sought after connection between cancer and cell cycle control.     

Theoretical calculations of the helix–coil transition of DNA in the presence of large,cooperatively binding ligands

Theoretical calculations are conducted on the helix–coil transition of DNA, in the presence of large, cooperatively binding ligands modeled after the DNA-binding proteins of current biological interest. The ligands are allowed to bind both to helx and to coil, to cover up any number of bases or base pairs in the complex, and to interact cooperatively with their nearest neighbors. The DNA is treated in the infinite homogeneous Ising model approximation, and all calculations are done by Lifson's method of sequence-generating functions. DNA melting curves are calculated by computer in order to expolore the effects on the transition of ligand size, binding constant, free activity, and ligand–ligand cooperativity. The calculations indicate that (1) at the same intrinsic free energy change per base pair of the complexes, small ligands, for purely entropic reasons, are more effective than are large ligands in shifting the DNA melting temperature; (2) the response of the DNA melting temperature to increased ligand binding constant K and/or free ligand activity L is adequately represented at high values of KL (but not at low KL) by a simple independent site model; (3) if curves are calculated with the total amount of added ligand remaining constant and the free ligand activity allowed to vary throughout the transition, biphasic melting curves can be obtained in the complete absence of ligand–ligand cooperativity. In an Appendix, the denaturation of poly[d(A-T)] in the presence of the drug, netropsin, is used to verify some features of the theory and to illustrate how the theory can be used to obtain numerical estimates of the ligand binding parameters from the experimental melting curves.     

Validation of measurements of ventilation-to-perfusion ratio inequality in the lung from expired gas.

The analysis of the gas in a single expirate has long been used to estimate the degree of ventilation-perfusion (Va/Q) inequality in the lung. To further validate this estimate, we examined three measures of Va/Q inhomogeneity calculated from a single full exhalation in nine anesthetized mongrel dogs under control conditions and after exposure to aerosolized methacholine. These measurements were then compared with arterial blood gases and with measurements of Va/Q inhomogeneity obtained using the multiple inert gas elimination technique. The slope of the instantaneous respiratory exchange ratio (R slope) vs. expired volume was poorly correlated with independent measures, probably because of the curvilinear nature of the relationship due to continuing gas exchange. When R was converted to the intrabreath Va/Q (iV/Q), the best index was the slope of iV/Q vs. volume over phase III (iV/Q slope). This was strongly correlated with independent measures, especially those relating to inhomogeneity of perfusion. The correlations for iV/Q slope and R slope considerably improved when only the first half of phase III was considered. We conclude that a useful noninvasive measurement of Va/Q inhomogeneity can be derived from the intrabreath respiratory exchange ratio.     

Ligand- and kinase activity-independent cell survival mediated by the epidermal growth factor receptor expressed in 32D cells

To investigate the intrinsic activities of the epidermal growth factor receptor and the role of its kinase domain in these functions within a cellular environment lacking endogenous ErbB protein expression, wild-type EGF receptor (WT-EGFR) and two kinase-impaired mutants, D813A and K721R, were expressed in 32D murine hematopoietic cells, a line which is normally dependent on interleukin 3 (IL3) for growth and survival. Addition of EGF in the absence of IL3 stimulates receptor autophosphorylation and, in the presence of serum, mitosis in cells expressing WT-EGFR, but not in cells expressing D813A or K721R. Unexpectedly, cells expressing WT-EGFR or K721R exhibited IL3-independent survival in the presence of fetal bovine serum; parental 32D cells and cells expressing D813A did not survive, apparently undergoing apoptosis in the absence of IL3, whether or not serum was present. Addition of EGF did not prevent the apoptosis of WT-EGFR or K721R cells in serum-free medium. Activation of Akt was not necessary to mediate the prosurvival activity of EGF receptor expression. These results suggest that the EGF receptor can mediate the prevention of apoptosis independently of both receptor-ligand binding and receptor kinase activity, and this activity is disrupted by the D813A mutation.     

Host response to malaria during pregnancy: placental monocyte recruitment is associated with elevated beta chemokine expression

Malaria during pregnancy is associated with poor birth outcomes, particularly low birth weight. Recently, monocyte infiltration into the placental intervillous space has been identified as a key risk factor for low birth weight. However, the malaria-induced chemokines involved in recruiting and activating placental monocytes have not been identified. In this study, we determined which chemokines are elevated during placental malaria infection and the association between chemokine expression and placental monocyte infiltration. Placental malaria infection was associated with elevations in mRNA expression of three beta chemokines, macrophage-inflammatory protein 1 (MIP-1) alpha (CCL3), monocyte chemoattractant protein 1 (MCP-1; CCL2), and I-309 (CCL1), and one alpha chemokine, IL-8 (CXCL8); all correlated with monocyte density in the placental intervillous space. Placental plasma concentrations of MIP-1 alpha and IL-8 were increased in women with placental malaria and were associated with placental monocyte infiltration. By immunohistochemistry, we localized placental chemokine production in malaria-infected placentas: some but not all hemozoin-laden maternal macrophages produced MIP-1 beta and MCP-1, and fetal stromal cells produced MCP-1. In sum, local placental production of chemokines is increased in malaria, and may be an important trigger for monocyte accumulation in the placenta.