Exosome-producing follicle associated epithelium is not involved in uptake of PrPd from the gut of sheep (Ovis aries): an ultrastructural study

In natural or experimental oral scrapie infection of sheep, disease associated prion protein (PrP(d)) often first accumulates in Peyer's patch (PP) follicles. The route by which infectivity reaches the follicles is unknown, however, intestinal epithelial cells may participate in intestinal antigenic presentation by delivering exosomes as vehicles of luminal antigens. In a previous study using an intestinal loop model, following inoculation of scrapie brain homogenate, inoculum associated PrP(d) was detected by light microscopy shortly (15 minutes to 3.5 hours) after inoculation in the villous lacteals and sub-mucosal lymphatics. No PrP(d) was located within the follicle-associated epithelium (FAE), sub-FAE domes or the PP follicles. To evaluate this gut loop model and the transportation routes in more detail, we used electron microscopy (EM) to study intestinal tissues exposed to scrapie or control homogenates for 15 minutes to 10 days. In addition, immuno-EM was used to investigate whether exosomes produced in the FAE may possess small amounts of PrP(d) that were not detectable by light microscopy. This study showed that the integrity of the intestinal epithelium was sustained in the intestinal loop model. Despite prominent transcytotic activity and exosome release from the FAE of the ileal PP in sheep, these structures were not associated with transportation of PrP(d) across the mucosa. The study did not determine how infectivity reaches the follicles of PPs. The possibility that the infectious agent is transported across the FAE remains a possibility if it occurs in a form that is undetectable by the methods used in this study. Infectivity may also be transported via lymph to the blood and further to all other lymphoid tissues including the PP follicles, but the early presence of PrP(d) in the PP follicles during scrapie infection argues against such a mechanism.     

Membrane insertion of the Escherichia coli MalF protein in cells with impaired secretion machinery

The MalF protein is an integral membrane protein of Escherichia coli containing eight membrane-spanning stretches and a large periplasmic domain of approximately 180 amino acids. We have asked whether this protein is dependent for its membrane insertion on the bacterial secretion machinery specified by the sec genes. Using azide to inhibit the SecA protein and sec mutants to reduce the functioning of the machinery, we have studied the membrane assembly of MalF and beta-galactosidase and alkaline phosphatase fusions to MalF. In no case did we see an effect of reducing sec gene function on the insertion of MalF or fusion proteins. Selection for mutants that would cause internalization of a MalF-beta-galactosidase hybrid protein yielded no mutations in sec genes. Our results suggest that MalF can assemble in the membrane independently of the bacterial secretion machinery.     

DnaB, DnaD and DnaI proteins are components of the Bacillus subtilis replication restart primosome.

Phenotypes of Bacillus subtilis priA mutants suggest that they are deficient in the restart of stalled chromosomal replication forks. The presumed activity of PriA in the restart process is to promote the assembly of a multiprotein complex, the primosome, which functions to recruit the replication fork helicase onto the DNA. We have proposed previously that three proteins involved in the initiation of replication at oriC in B. subtilis, DnaB, DnaD and DnaI, are components of the PriA primosome in this bacterium. However, the involvement of these proteins in replication restart has not yet been studied. Here, we describe dnaB mutations that suppress the phenotypes of B. subtilis priA mutants. In a representative mutant, the DnaC helicase is loaded onto single-stranded DNA in a PriA-independent, DnaD- and DnaI-dependent manner. These observations confirm that DnaB, DnaD and DnaI are primosomal proteins in B. subtilis. Moreover, their involvement in the suppression of priA phenotypes shows that they participate in replication fork restart in B. subtilis.     

Haptoglobin and CCR2 receptor expression in ovarian cancer cells that were exposed to ascitic fluid: Exploring a new role of haptoglobin in the tumoral microenvironment

Haptoglobin (Hp) is an acute-phase protein that is produced by the liver to capture the iron that is present in the blood circulation, thus avoiding its accumulation in the blood. Moreover, Hp has been detected in a wide variety of tissues, in which it performs various functions. In addition, this protein is considered a potential biomarker in many diseases, such as cancer, including ovarian carcinoma; however, its participation in the cancerous processes has not yet been determined. The objective of this work was to demonstrate the expression of Hp and its receptor CCR2 in the ovarian cancer cells and its possible involvement in the process of cell migration through changes in the rearrangement of the actin cytoskeleton using western blot and wound-healing assays and confirming by confocal microscopy. Ovarian cancer cells express both Hp and its receptor CCR2 but only after exposure to ascitic fluid, inducing moderated cell migration. However, when the cells are exposed to exogenous Hp, the expression of CCR2 is induced together with drastic changes in the actin cytoskeleton rearrangement. At the same time, Hp induced cell migration in a much more efficient manner than did ascitic fluid. These effects were blocked when the CCR2 synthetic antagonist RS102895 was used to pretreat the cells. These results suggest that Hp-induced changes in the cell morphology, actin cytoskeleton structure, and migration ability of tumor cells, is possibly “preparing” these cells for the potential induction of the metastatic phenotype.     

Quality improvement report: The “jaundice hotline” for the rapid assessment of patients with jaundice

ProblemPatients with jaundice require rapid diagnosis and treatment, yet such patients are often subject to delay.DesignAn open referral, rapid access jaundice clinic was established by reorganisation of existing services and without the need for significant extra resources.

Background and setting

A large general hospital in a largely rural and geographically isolated area.

Key measures for improvement

Waiting times for referral, consultation, diagnosis, and treatment, length of stay in hospital, and general practitioners'' and patients'' satisfaction with the service.

Strategies for change

Referrals were made through a 24 hour telephone answering machine and fax line. Initial assessment of patients was carried out by junior staff as part of their working week. Dedicated ultrasonography appointments were made available.

Effects of change

Of 107 patients seen in the first year of the service, 62 had biliary obstruction. The mean time between referral and consultation was 2.5 days. Patients who went on to endoscopic retrograde cholangiopancreatography waited 5.7 days on average. The mean length of stay in hospital in the 69 patients who were admitted was 6.1 days, compared with 11.5 days in 1996, as shown by audit data. Nearly all the 36 general practices (95%) and the 30 consecutive patients (97%) that were surveyed rated the service as above average or excellent.

Lessons learnt

An open referral, rapid access service for patients with jaundice can shorten time to diagnosis and treatment and length of stay in hospital. These improvements can occur through the reorganisation of existing services and with minimal extra cost.     

A versatile oxygenator and perfusion system for magnetic resonance studies

A compact, reusable membrane oxygenator has been constructed for the perfusion of cultured cells and isolated organs. While the oxygenator was designed to be compatible with nuclear magnetic resonance (NMR) spectroscopy studies, it can also be used for any experiment which requires warming and oxygenation of perfusates. For the NMR studies, the oxygenator can be positioned at the opening of the magnet bore which allows oxygenation and warming of the perfusate immediately prior to delivery to the tissue, therefore eliminating problems with heat or oxygen loss which may occur with the long perfusion lines. (c) 1996 John Wiley & Sons, Inc.     

Effects of endothelin and relaxin on rat uterine segment contractility.

In order to determine the effects of endothelin (ET) and relaxin on uterine contractility, immature female rats were treated with estrogen (E, 1 microgram s.c., Days 1-3) or estrogen and progesterone (2 mg s.c. [E + P], Days 2 and 3), and killed; the uterine horns were removed and suspended in muscle baths. Initially, we determined the contractile response to varying doses of ET and how this response was altered by pretreatment with progesterone. Uterine strips from animals treated with E + P (n = 10) were less sensitive to the stimulatory effects of ET than were strips from E-treated animals (n = 10). This difference was significant at ET doses above 2.5 nM. After completion of the dose-response studies, contractile patterns in response to ET and relaxin were then studied in animals treated with E (n = 10) or E + P (n = 9). ET (5 nM) significantly increased uterine contractility, mostly through an effect on the frequency of contractions (p less than 0.01). Relaxin (25 ng/ml) decreased contractility, affecting all contractile parameters measured (p less than 0.01). ET stimulated contractility in uterine horn segments previously inhibited by relaxin (p less than 0.01), and relaxin reduced the increased contractility produced by earlier exposure to ET (p less than 0.01). These data indicate that ET and relaxin can interact reversibly to control contractility in uterine horn segments in vitro, and that progesterone pretreatment can diminish the contractile response to the stimulatory effects of ET.     

Sterilization of boll weevil pupae with fractionated doses of gamma irradiation

Fractionated doses of 6,250–8,000 rads of gamma irradiation when administered to pupae of the boll weevil, Anthonomus grandis Boh., sexually sterilized both sexes. Mortality of males thus treated with 6,250 and 8,000 rads via fractionation was 14% and 27% respectively, by 5 days posttreatment compared with 46% mortality when an equivalent acute dose was administered to newly emerged adults. Pheromone production of males irradiated at 6,250 rads was one-third that of the control for the first 4 days, but equal that of the control during 5–11 days posttreatment. This procedure lends itself to the large-scale sterilization of weevils needed in an eradication program. This technique is applicable to other insects that are highly susceptible to acute doses.

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Zusammenfassung Durch fraktionierte Dosen von insgesamt 6250 und 8000 rads einer Gammabestrahlung (25 bzw. 32 Teilbehandlungen zu 250 rads mit 4 h-Intervallen) wurden Puppen des Baumwollkapselkäfers, Anthonomus grandis Boheman, in beiden Geschlechtern sexuell sterilisiert. Die Sterblichkeit, 5 d nach der Behandlung, betrug bei den so behandelten Männchen bei 6250 rads 14%, bei 8000 rads 27% im Vergleich zu 46%, wenn eine äquivalente akute Dosis bei frisch geschlüpften Käfern angewendet wurde. Die Pheromonproduktion der Männchen in den ersten 4 d erreichte bei 6250 rads-Bestrahlung 1/3 derjenigen der Kontrolltiere; 5–11 d nach der Behandlung war kein Unterschied. Dieses Verfahren eignet sich für die Massensterilisation von Kapselkäfern, wie sie bei einem Ausrottungsprogramm nötig ist. Diese Technik ist anwendbar auf andere Insekten, die gegenüber akuten Dosen hochempfindlich sind.

     

Functional interplay between the Bacillus subtilis DnaD and DnaB proteins essential for initiation and re-initiation of DNA replication

Initiation and re-initiation of chromosomal DNA replication in bacteria rely on divergent multiprotein assemblies, which direct the functional delivery of the replicative helicase on single-stranded DNA (ssDNA) at specific sites. These two processes are triggered either at the single chromosomal origin oriC or at arrested forks by the conserved DnaA and PriA proteins respectively. In Bacillus subtilis, these two pathways further require the three essential proteins DnaB, DnaD and DnaI, restrictively encoded in Gram positive bacteria of low GC content. We have recently shown that DnaI and DnaB act as a pair of loaders of the DnaC replicative helicase. The role of DnaD appeared more enigmatic. It was previously shown to interact with DnaA and to display weak ssDNA binding activity. Here, we report that purified DnaD can interact physically with PriA and with DnaB. We show that the lethality of the temperature-sensitive dnaD23 mutant can be suppressed by different DnaB point mutants, which were found to be identical to the suppressors of priA null mutants. The DnaD23 protein displays lower ssDNA binding activity than DnaD. Conversely, the DnaB75 protein, the main dnaD23 suppressor, has gained affinity for ssDNA. Finally, we observed that this interplay between DnaD and DnaB is crucial for their concerted interaction with SSB-coated ssDNA, which is the expected substrate for the loading of the replicative helicase in vivo. Altogether, these results highlight the need for both DnaD and DnaB to interact individually and together with ssDNA during the early stages of initiation and re-initiation of chromosomal DNA replication. They also point at a main structural role of DnaD in the multiprotein assemblies built during these two essential processes.     

Range contraction and increasing isolation of a polar bear subpopulation in an era of sea‐ice loss

Climate change is expected to result in range shifts and habitat fragmentation for many species. In the Arctic, loss of sea ice will reduce barriers to dispersal or eliminate movement corridors, resulting in increased connectivity or geographic isolation with sweeping implications for conservation. We used satellite telemetry, data from individually marked animals (research and harvest), and microsatellite genetic data to examine changes in geographic range, emigration, and interpopulation connectivity of the Baffin Bay (BB) polar bear (Ursus maritimus) subpopulation over a 25‐year period of sea‐ice loss. Satellite telemetry collected from n = 43 (1991–1995) and 38 (2009–2015) adult females revealed a significant contraction in subpopulation range size (95% bivariate normal kernel range) in most months and seasons, with the most marked reduction being a 70% decline in summer from 716,000 km² (SE 58,000) to 211,000 km² (SE 23,000) (p < .001). Between the 1990s and 2000s, there was a significant shift northward during the on‐ice seasons (2.6^° shift in winter median latitude, 1.1^° shift in spring median latitude) and a significant range contraction in the ice‐free summers. Bears in the 2000s were less likely to leave BB, with significant reductions in the numbers of bears moving into Davis Strait (DS) in winter and Lancaster Sound (LS) in summer. Harvest recoveries suggested both short and long‐term fidelity to BB remained high over both periods (83–99% of marked bears remained in BB). Genetic analyses using eight polymorphic microsatellites confirmed a previously documented differentiation between BB, DS, and LS; yet weakly differentiated BB from Kane Basin (KB) for the first time. Our results provide the first multiple lines of evidence for an increasingly geographically and functionally isolated subpopulation of polar bears in the context of long‐term sea‐ice loss. This may be indicative of future patterns for other polar bear subpopulations under climate change.