ApoC-III content of apoB-containing lipoproteins is associated with binding to the vascular proteoglycan biglycan

Retention of apolipoprotein (apo)B and apoE-containing lipoproteins by extracellular vascular proteoglycans is critical in atherogenesis. Moreover, high circulating apoC-III levels are associated with increased atherosclerosis risk. To test whether apoC-III content of apoB-containing lipoproteins affects their ability to bind to the vascular proteoglycan biglycan, we evaluated the impact of apoC-III on the interaction of [(35)S]SO(4)-biglycan derived from cultured arterial smooth muscle cells with lipoproteins obtained from individuals across a spectrum of lipid concentrations. The extent of biglycan binding correlated positively with apoC-III levels within VLDL (r = 0.78, P < 0.01), IDL (r = 0.67, P < 0.01), and LDL (r = 0.52, P < 0.05). Moreover, the biglycan binding of VLDL, IDL, and LDL was reduced after depletion of apoC-III-containing lipoprotein particles in plasma by anti-apoC-III immunoaffinity chromatography. Since apoC-III does not bind biglycan directly, enhanced biglycan binding may result from a conformational change associated with increased apo C-III content by which apoB and/or apoE become more accessible to proteoglycans. This may be an intrinsic property of lipoproteins, since exogenous apoC-III enrichment of LDL and VLDL did not increase binding. ApoC-III content may thus be a marker for lipoproteins characterized as having an increased ability to bind proteoglycans.     

Dynamic regulation of HIV-1 mRNA populations analyzed by single-molecule enrichment and long-read sequencing

Alternative RNA splicing greatly expands the repertoire of proteins encoded by genomes. Next-generation sequencing (NGS) is attractive for studying alternative splicing because of the efficiency and low cost per base, but short reads typical of NGS only report mRNA fragments containing one or few splice junctions. Here, we used single-molecule amplification and long-read sequencing to study the HIV-1 provirus, which is only 9700 bp in length, but encodes nine major proteins via alternative splicing. Our data showed that the clinical isolate HIV-1_89.6 produces at least 109 different spliced RNAs, including a previously unappreciated ∼1 kb class of messages, two of which encode new proteins. HIV-1 message populations differed between cell types, longitudinally during infection, and among T cells from different human donors. These findings open a new window on a little studied aspect of HIV-1 replication, suggest therapeutic opportunities and provide advanced tools for the study of alternative splicing.     

Equilibrium and metastable states in lecithin films.

We have considered whether lecithin surface films below the gel-liquid crystal transition temperature, Tc, are in unique physical states. In general, below Tc, equilibrium films do not exist when surface pressures, pi, exceed about 0.1 dyn/cm. Since surface pressure-surface area isotherms of lecithin films below Tc always encompass pi's much greater than 0.1 dyn/cm, the film states are metastable. We show that the film properties under these conditions depend strongly on the history of the film, particularly the method of film formation. Lecithin surface films below Tc are thus in arbitrary metastable states, so that pi-area isotherms are difficult to interpret. The physical significance of such isotherms remains to be determined. The utility of pure lecithin surface layers below Tc as models for biological systems is also challenged by our results.     

Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation

The C-terminal Src kinase (Csk) phosphorylates and down-regulates Src family tyrosine kinases. The Csk-binding protein (Cbp) localizes Csk close to its substrates at the plasma membrane, and increases the specific activity of the kinase. To investigate this long-range catalytic effect, the phosphorylation of Src and the conformation of Csk were investigated in the presence of a high-affinity phosphopeptide derived from Cbp. This peptide binds tightly to the SH2 domain and enhances Src recognition (lowers K(m)) by increasing the apparent phosphoryl transfer rate in the Csk active site, a phenomenon detected in rapid quench flow experiments. Previous studies demonstrated that the regulation of Csk activity is linked to conformational changes in the enzyme that can be probed with hydrogen-deuterium exchange methods. We show that the Cbp peptide impacts deuterium incorporation into its binding partner (the SH2 domain), and into the SH2-kinase linker and several sequences in the kinase domain, including the glycine-rich loop in the active site. These findings, along with computational data from normal mode analyses, suggest that the SH2 domain moves in a cantilever fashion with respect to the small lobe of the kinase domain, ordering the active site for catalysis. The binding of a small Cbp-derived peptide to the SH2 domain of Csk modifies these motions, enhancing Src recognition.     

Precipitation-dependent flowering of Globularia alypum and Erica multiflora in Mediterranean shrubland under experimental drought and warming, and its inter-annual variability

Background and Aims

Relationships between autumn flowering, precipitation and temperature of plant species of Mediterranean coastal shrublands have been described, but not analysed experimentally. These relationships were analysed for two species of co-occurring, dominant, autumn-flowering shrubs, Globularia alypum and Erica multiflora, over 4 years and in experimentally generated drought and warming conditions. The aim was to improve predictions about the responses and adaptations of flowering of Mediterranean vegetation to climate change.

Methods

Beginning of anthesis and date of maximum flowering intensity (‘peak date’) were monitored over 4 years (2001–2004) on a garrigue land type in the noth-east of the Iberian Peninsula. Two experimental treatments were applied, increased temperature (+0·73°C) and reduced soil moisture (–17%) relative to untreated plots.

Key Results

Flowering of Globularia alypum and Erica multiflora differed greatly between years depending on the precipitation of the previous months and the date of the last substantial rainfall (>10 mm). Globularia alypum flowered once or twice (unimodal or bimodal) as the result of differences in the distribution and magnitude of precipitation in late-spring and summer (when floral buds develop). The drought treatment delayed and decreased flowering of Globularia alypum in 2001 and delayed flowering in 2002. Warming extended the period between the beginning of flowering and the end of the second peak for autumn flowering in 2001 and also increased peak intensity in 2002. Flowering of Erica multiflora was unaffected by either treatment.

Conclusions

Autumn flowering of Globularia alypum and Erica multiflora is more dependent on water availability than on temperature. Considerable inter-annual plasticity in the beginning of anthesis and peak date and on unimodal or bimodal flowering constitutes a ‘safe strategy’ for both species in relation to varying precipitation and temperature. However, severe changes in precipitation in spring and summer may severely affect flowering of Globularia alypum but not Erica multiflora, thus affecting development/structure of the ecosystem if such conditions persist.Key words: Globularia alypum, Erica multiflora, autumn flowering, drought, global warming, Mediterranean     

Hypohidrotic ectodermal dysplasia

Summary A family carrying the X-linked gene for hypohidrotic ectodermal dysplasia (hereditary ectodermal polydysplasia or Christ-Siemens-Touraine syndrome) over three generations was monitored for more than 15 years. Two prenatal diagnoses were carried out by fetoscopy on skin biopsies. Polymorphic probes were used in the segregation analysis of the Xq11–21 region carried out on 30 members of the family. Current screening possiblitities for the carriers and prenatal diagnosis are discussed.     

Haplotypes of the bovine IgG2 heavy gamma chain in tick-resistant and tick-susceptible breeds of cattle

Bovines present contrasting, heritable phenotypes of infestations with the cattle tick, Rhipicephalus (Boophilus) microplus. Tick salivary glands produce IgG-binding proteins (IGBPs) as a mechanism for escaping from host antibodies that these ectoparasites ingest during blood meals. Allotypes that occur in the constant region of IgG may differ in their capacity to bind with tick IGBPs; this may be reflected by the distribution of distinct allotypes according to phenotypes of tick infestations. In order to test this hypothesis, we investigated the frequency of haplotypes of bovine IgG2 among tick-resistant and tick-susceptible breeds of bovines. Sequencing of the gene coding for the heavy chain of IgG2 from 114 tick-resistant (Bos taurus indicus, Nelore breed) and tick-susceptible (B. t. taurus, Holstein breed) bovines revealed SNPs that generated 13 different haplotypes, of which 11 were novel and 5 were exclusive of Holstein and 3 of Nelore breeds. Alignment and modeling of coded haplotypes for hinge regions of the bovine IgG2 showed that they differ in the distribution of polar and hydrophobic amino acids and in shape according to the distribution of these amino acids. We also found that there was an association between genotypes of the constant region of the IgG2 heavy chain with phenotypes of tick infestations. These findings open the possibility of investigating if certain IgG allotypes hinder the function of tick IGBPs. If so, they may be markers for breeding for resistance against tick infestations.     

Sorghum Phytochrome B Inhibits Flowering in Long Days by Activating Expression of SbPRR37 and SbGHD7, Repressors of SbEHD1, SbCN8 and SbCN12

Light signaling by phytochrome B in long days inhibits flowering in sorghum by increasing expression of the long day floral repressors PSEUDORESPONSE REGULATOR PROTEIN (SbPRR37, Ma1) and GRAIN NUMBER, PLANT HEIGHT AND HEADING DATE 7 (SbGHD7, Ma6). SbPRR37 and SbGHD7 RNA abundance peaks in the morning and in the evening of long days through coordinate regulation by light and output from the circadian clock. 58 M, a phytochrome B deficient (phyB-1, ma3^R) genotype, flowered ∼60 days earlier than 100 M (PHYB, Ma3) in long days and ∼11 days earlier in short days. Populations derived from 58 M (Ma1, ma3^R, Ma5, ma6) and R.07007 (Ma1, Ma3, ma5, Ma6) varied in flowering time due to QTL aligned to PHYB/phyB-1 (Ma3), Ma5, and GHD7/ghd7-1 (Ma6). PHYC was proposed as a candidate gene for Ma5 based on alignment and allelic variation. PHYB and Ma5 (PHYC) were epistatic to Ma1 and Ma6 and progeny recessive for either gene flowered early in long days. Light signaling mediated by PhyB was required for high expression of the floral repressors SbPRR37 and SbGHD7 during the evening of long days. In 100 M (PHYB) the floral activators SbEHD1, SbCN8 and SbCN12 were repressed in long days and de-repressed in short days. In 58 M (phyB-1) these genes were highly expressed in long and short days. Furthermore, SbCN15, the ortholog of rice Hd3a (FT), is expressed at low levels in 100 M but at high levels in 58 M (phyB-1) regardless of day length, indicating that PhyB regulation of SbCN15 expression may modify flowering time in a photoperiod-insensitive manner.     

Sexual differentiation in quail: Conversion of androgen to estrogen mediates testosterone-induced demasculinization of copulation but not other male characteristics

Previous research has shown that administration of either testosterone or estradiol to male quail embryos will demasculinize behavior and morphology. Six experiments in which embryos were treated were conducted to test the hypothesis that this testosterone-induced demasculinization is due to conversion of testosterone to estrogen (aromatization). In Experiment 1, dihydrotestosterone propionate, a nonaromatizable androgen, failed to demasculinize copulatory behavior, but did demasculinize crowing, strutting, and proctodeal glands. In Experiment 2, injection of the aromatizable androgens testosterone propionate (TP), testosterone, or androstenedione demasculinized copulatory behavior, the nonaromatizable androgen androsterone failed to have such an effect, and all androgens demasculinized proctodeal glands. In Experiment 3, Silastic implants of testosterone demasculinized all male characteristics, whereas implants of androsterone demasculinized only proctodeal glands. In Experiment 4, the antiestrogen tamoxifen prevented TP from demasculinizing copulatory behavior, but had no such effect with respect to crowing and strutting. In Experiments 5 and 6, the aromatization inhibitor 1,4,6-androstatrien-3,17-dione (ATD) prevented TP but not estradiol benzoate from demasculinizing copulatory behavior. Thus (1) in quail, testosterone-induced demasculinization of copulatory behavior is due to androgen aromatization, whereas testosterone-induced demasculinization of crowing, strutting, and proctodeal glands is not; (2) the distinct components of normal male reproductive behavior exhibit different patterns of steroid specificity during the organizational period, as was previously shown for the activational period; (3) the steroid specificity of crowing, strutting, and proctodeal glands changes between the organizational and activational periods. During organization, there is little specificity, whereas during activation, these characteristics respond only to androgens, never to estrogens. This difference suggests that developmental changes have occurred in the underlying biochemical substrates.