Protein-disulfide isomerase displaces the cholera toxin A1 subunit from the holotoxin without unfolding the A1 subunit

Protein-disulfide isomerase (PDI) has been proposed to exhibit an "unfoldase" activity against the catalytic A1 subunit of cholera toxin (CT). Unfolding of the CTA1 subunit is thought to displace it from the CT holotoxin and to prepare it for translocation to the cytosol. To date, the unfoldase activity of PDI has not been demonstrated for any substrate other than CTA1. An alternative explanation for the putative unfoldase activity of PDI has been suggested by recent structural studies demonstrating that CTA1 will unfold spontaneously upon its separation from the holotoxin at physiological temperature. Thus, PDI may simply dislodge CTA1 from the CT holotoxin without unfolding the CTA1 subunit. To evaluate the role of PDI in CT disassembly and CTA1 unfolding, we utilized a real-time assay to monitor the PDI-mediated separation of CTA1 from the CT holotoxin and directly examined the impact of PDI binding on CTA1 structure by isotope-edited Fourier transform infrared spectroscopy. Our collective data demonstrate that PDI is required for disassembly of the CT holotoxin but does not unfold the CTA1 subunit, thus uncovering a new mechanism for CTA1 dissociation from its holotoxin.     

ADP‐ribosylation factor 6 acts as an allosteric activator for the folded but not disordered cholera toxin A1 polypeptide

The catalytic A1 subunit of cholera toxin (CTA1) has a disordered structure at 37°C. An interaction with host factors must therefore place CTA1 in a folded conformation for the modification of its Gsα target which resides in a lipid raft environment. Host ADP‐ribosylation factors (ARFs) act as in vitro allosteric activators of CTA1, but the molecular events of this process are not fully characterized. Isotope‐edited Fourier transform infrared spectroscopy monitored ARF6‐induced structural changes to CTA1, which were correlated to changes in CTA1 activity. We found ARF6 prevents the thermal disordering of structured CTA1 and stimulates the activity of stabilized CTA1 over a range of temperatures. Yet ARF6 alone did not promote the refolding of disordered CTA1 to an active state. Instead, lipid rafts shifted disordered CTA1 to a folded conformation with a basal level of activity that could be further stimulated by ARF6. Thus, ARF alone is unable to activate disordered CTA1 at physiological temperature: additional host factors such as lipid rafts place CTA1 in the folded conformation required for its ARF‐mediated activation. Interaction with ARF is required for in vivo toxin activity, as enzymatically active CTA1 mutants that cannot be further stimulated by ARF6 fail to intoxicate cultured cells.     

Passage of molecules across the intercellular bridge between post-mitotic daughter cells

Living mitotic HeLa cells were microinjected with sodium fluorescein, fluorescein isothiocyanate (FITC)-labelled IgG, and Lucifer Yellow-CH to determine if the intracellular bridge and midbody forms a barrier to the migration of these molecules. All three fluorescent molecules were found to pass across the intercellular bridge. The post-mitotic cells maintained a molecular patency across the intercellular bridge that persisted for considerable lengths of time, extending into the next cell cycle.     

Regional expression of prostaglandin E2 and F2alpha receptors in human myometrium, amnion, and choriodecidua with advancing gestation and labor

The change from uterine quiescence to enhanced contractile activity may be due to the differential expression of prostaglandin receptors within the myometrium and fetal membranes, in a temporal and topographically distinct manner. To address this question, we determined the localization and expression of the PGE2 receptor subtypes (PTGER1-4) and the PGF2alpha receptor (PTGFR) in paired upper and lower segment myometrium, amnion, and choriodecidual samples throughout human pregnancy, with and without labor. All receptor subtypes were found throughout the muscle layers in both the upper and lower uterine segments, colocalizing with alpha smooth muscle actin. A change in intracellular localization was observed at term labor, where PTGER1 and PTGER4 were predominately associated with the nucleus. Minimal changes in the expression of the PGE2 and PGF2alpha receptor subtypes were observed with gestational age, labor, or between the upper and lower myometrial segments. Receptor expression in maternal and fetal tissues differed between the receptor subtypes; PTGER1 and PTGER4 were predominately expressed in the fetal membranes, PTGER2 was greatest in the myometrium, whereas PTGER3 and PTGFR were similarly expressed in the myometrium and fetal membranes. Myometrial activation through the prostaglandin receptors is perhaps more subtle and may be mediated by a balance between one or several of the prostaglandin receptor subtypes together with other known contraction associated proteins. Lack of coordination in receptor expression between the myometrium and fetal membranes may indicate different regulatory mechanisms between these tissues, or it may suggest a function for these receptors in the amnion and choriodecidua that is independent of that seen in the myometrium.     

Interactions between inflammatory signals and the progesterone receptor in regulating gene expression in pregnant human uterine myocytes

The absence of a fall in circulating progesterone levels has led to the concept that human labour is associated with 'functional progesterone withdrawal' caused through changes in the expression or function of progesterone receptor (PR). At the time of labour, the human uterus is heavily infiltrated with inflammatory cells, which release cytokines to create a 'myometrial inflammation' via NF-κB activation. The negative interaction between NF-κB and PR, may represent a mechanism to account for 'functional progesterone withdrawal' at term. Conversely, PR may act to inhibit NF-κB function and so play a role in inhibition of myometrial inflammation during pregnancy. To model this inter-relationship, we have used small interfering (si) RNA-mediated knock-down of PR in human pregnant myocytes and whole genome microarray analysis to identify genes regulated through PR. We then activated myometrial inflammation using IL-1β stimulation to determine the role of PR in myometrial inflammation regulation. Through PR-knock-down, we found that PR regulates gene networks involved in myometrial quiescence and extracellular matrix integrity. Activation of myometrial inflammation was found to antagonize PR-induced gene expression, of genes normally upregulated via PR. We found that PR does not play a role in repression of pro-inflammatory gene networks induced by IL-1β and that only MMP10 was significantly regulated in opposite directions by IL-1β and PR. We conclude that progesterone acting through PR does not generally inhibit myometrial inflammation. Activation of myometrial inflammation does cause 'functional progesterone withdrawal' but only in the context of genes normally upregulated via PR.     

Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp. AN-7

A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch–peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 °C and 6.0. With half-life time longer than one day at 80 °C the enzyme proves to be thermostable in the pH range 4.5–7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 °C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg²⁺ ions. Ca²⁺, dithiothreitol, and Mn²⁺ stimulated the pullulanase activity. Kinetic experiments at 80 °C and pH 6.0 gave V_max and K_m values of 154 U mg⁻¹ and 1.3 mg ml⁻¹. The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. To our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date.     

Improved bioethanol production through simultaneous saccharification and fermentation of lignocellulosic agricultural wastes by <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> 6556

The combined effect of simultaneous saccharification and fermentation and separate hydrolysis and fermentation (SHF) for ethanol production by Kluyveromyces marxianus 6556 was studied using two lignocellulosic feedstocks viz., corncob and soybean cake. The ethanologenic efficiency of K. marxianus 6556 was observed as 28% (theoretical yield) in a fermentation medium containing glucose, but, there was no ethanol production by cells grown on xylose. A maximum sugar release of 888 mg/g corncob and 552 mg/g soybean cake was achieved through acid hydrolysis pretreatment. Furthermore, corncob and soybean cake treated with commercial cellulase (100 IU for 48 h) from Trichoderma reesei yielded reducing sugars of 205 and 100 mg/g, respectively. Simultaneous saccharification and fermentation resulted in highest ethanol production of 5.68 g/l on corncob and 2.14 g/l on soybean cake after 48 h of incubation. On the contrary, the presence of inhibitors decreased the overall ethanol yield in the hydrolysates obtained through SHF of corncob and soybean cake.     

A dual origin of the Xist gene from a protein-coding gene and a set of transposable elements

X-chromosome inactivation, which occurs in female eutherian mammals is controlled by a complex X-linked locus termed the X-inactivation center (XIC). Previously it was proposed that genes of the XIC evolved, at least in part, as a result of pseudogenization of protein-coding genes. In this study we show that the key XIC gene Xist, which displays fragmentary homology to a protein-coding gene Lnx3, emerged de novo in early eutherians by integration of mobile elements which gave rise to simple tandem repeats. The Xist gene promoter region and four out of ten exons found in eutherians retain homology to exons of the Lnx3 gene. The remaining six Xist exons including those with simple tandem repeats detectable in their structure have similarity to different transposable elements. Integration of mobile elements into Xist accompanies the overall evolution of the gene and presumably continues in contemporary eutherian species. Additionally we showed that the combination of remnants of protein-coding sequences and mobile elements is not unique to the Xist gene and is found in other XIC genes producing non-coding nuclear RNA.     

Geographic distributions and origins of human head lice (Pediculus humanus capitis) based on mitochondrial data

Human head lice (Pediculus humanus capitis) are subdivided into 3 deeply divergent mitochondrial clades (Clades A, B, and C), each having unique geographical distributions. Determining the evolutionary history and geographic distribution of these mitochondrial clades can elucidate the evolutionary history of the lice as well as their human hosts. Previous data suggest that lice belonging to mitochondrial Clade B may have originated in North America or Asia; however, geographic sampling and sample sizes have been limited. With newly collected lice, we calculate the relative frequency, geographic distribution, and genetic diversity of louse mitochondrial clades to determine the geographic origin of lice belonging to Clade B. In agreement with previous studies, genetic diversity data support a North American origin of Clade B lice. It is likely that lice belonging to this mitochondrial clade recently migrated to other geographic localities, e.g., Europe and Australia, and, if not already present, may disperse further to occupy all geographic regions.