Structure and possible functions of the calcospherite-rich cells (R* cells) in the digestive gland of the shore crab CArcinus maenas

Summary R^*-cells of the digestive gland of Carcinus maenas have been investigated functionally and morphologically. A comparison of the capacity of separated cell suspensions to synthesize glycogen gave support to the hypothesis that R and R^* cells belong to the same cell line. The unexpected observation of R^* cells in gastric juice suggests that their release could represent a mode of redistribution of carbohydrate stores when the feeding activity of the crab is lower. Under electron microscopy, the calcospherites of R^* cells appeared to be surrounded by multiple membranous layers, and displayed tubular and vesicular structures in their core. High glucose-6-phosphatase (G6Pase) activity in the subcellular fraction that is enriched in calcospherites suggests that these membranes are derived from the endoplasmic reticulum, via a process in which the enzyme plays a key role. We propose that this is the way by which the R cell differentiates into R^* cell.     

Female sperm use and storage between fertilization events drive sperm competition and male ejaculate allocation

Sperm competition theory has traditionally focused on how male allocation responds to female promiscuity, when males compete to fertilize a single clutch of eggs. Here, we develop a model to ask how female sperm use and storage across consecutive reproductive events affect male ejaculate allocation and patterns of mating and paternity. In our model, sperm use (a single parameter under female control) is the main determinant of sperm competition, which alters the effect of female promiscuity on male success and, ultimately, male reproductive allocation. Our theory reproduces the general pattern predicted by existing theory that increased sperm competition favors increased allocation to ejaculates. However, our model predicts a negative correlation between male ejaculate allocation and female promiscuity, challenging the generality of a prevailing expectation of sperm competition theory. Early models assumed that the energetic costs of precopulatory competition and the level of sperm competition are both determined by female promiscuity, which leads to an assumed covariation between these two processes. By modeling precopulatory costs and sperm competition independently, our theoretical framework allows us to examine how male allocation should respond independently to variation in sperm competition and energetic trade‐offs in mating systems that have been overlooked in the past.     

The rosettazyme: A synthetic cellulosome

Cellulose is an attractive feedstock for biofuel production because of its abundance, but the cellulose polymer is extremely stable and its constituent sugars are difficult to access. In nature, extracellular multi-enzyme complexes known as cellulosomes are among the most effective ways to transform cellulose to useable sugars. Cellulosomes consist of a diversity of secreted cellulases and other plant cell-wall degrading enzymes bound to a protein scaffold. These scaffold proteins have cohesin modules that bind conserved dockerin modules on the enzymes. It is thought that the localization of these diverse enzymes on the scaffold allows them to function synergistically. In order to understand and harness this synergy smaller, simplified cellulosomes have been constructed, expressed, and reconstituted using truncated cohesin-containing scaffolds.Here we show that an 18-subunit protein complex called a rosettasome can be genetically engineered to bind dockerin-containing enzymes and function like a cellulosome. Rosettasomes are thermostable, group II chaperonins from the hyperthermo-acidophilic archaeon Sulfolobus shibatae, which in the presence of ATP/Mg²⁺ assemble into 18-subunit, double-ring structures. We fused a cohesin module from Clostridium thermocellum to a circular permutant of a rosettasome subunit, and we demonstrate that the cohesin–rosettasomes: (1) bind dockerin-containing endo- and exo-gluconases, (2) the bound enzymes have increased cellulose-degrading activity compared to their activity free in solution, and (3) this increased activity depends on the number and ratio of the bound glucanases. We call these engineered multi-enzyme structures rosettazymes.     

Activation of cardiac AMP-activated protein kinase by LKB1 expression or chemical hypoxia is blunted by increased Akt activity

AMP-activated protein kinase (AMPK) plays a major role in the regulation of cardiac energy substrate utilization and can be negatively regulated by Akt activation in the heart. It has recently been shown that Akt directly phosphorylates AMPKalpha(1)/alpha(2) on Ser(485/491) in vitro and prevents the AMPK kinase (AMPKK) LKB1 from phosphorylating AMPKalpha at its primary activation site, Thr(172) (S Horman, D Vertommen, R Heath, D Neumann, V Mouton, A Woods, U Schlattner, T Wallimann, D Carling, L Hue, and MH Rider. J Biol Chem 281: 5335-5340, 2006). To determine whether this is also the case in the cardiac myocyte, neonatal rat cardiac myocytes (NRCM) were infected with a recombinant adenovirus expressing a constitutively active mutant of Akt1 (myrAkt1) and then with or without adenoviruses expressing the active LKB1 complex. Expression of myrAkt1 blunted LKB1-induced phosphorylation of AMPKalpha at Thr(172), which resulted in a dramatic decrease in phosphorylation of AMPK's target, acetyl CoA-carboxylase. This decrease in AMPK activity was associated with prior Akt1-dependent phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491). To investigate whether Akt1 activation was also able to prevent other AMPKKs from phosphorylating AMPKalpha, we subjected NRCM to chemical hypoxia and noted a marked increase in phosphorylation of AMPKalpha at Thr(172), despite no change in LKB1 activity. NRCM expressing myrAkt1 demonstrated increased phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491) and a complete inhibition of chemical hypoxia-induced phosphorylation of AMPKalpha at Thr(172). Taken together, our data show that activation of Akt1 is able to prevent activation of cardiac AMPK by LKB1 and at least one other AMPKK, likely by prior phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491).     

Opt-Out Panel Testing for HIV,Hepatitis B and Hepatitis C in an Urban Emergency Department: A Pilot Study

Objectives

Studies suggest 2 per 1000 people in Dublin are living with HIV, the level above which universal screening is advised. We aimed to assess the feasibility and acceptability of a universal opt-out HIV, Hepatitis B and Hepatitis C testing programme for Emergency Department patients and to describe the incidence and prevalence of blood-borne viruses in this population.

Methods

An opt-out ED blood borne virus screening programme was piloted from March 2014 to January 2015. Patients undergoing blood sampling during routine clinical care were offered HIV 1&2 antibody/antigen assay, HBV surface antigen and HCV antibody tests. Linkage to care where necessary was co-ordinated by the study team. New diagnosis and prevalence rates were defined as the new cases per 1000 tested and number of positive tests per 1000 tested respectively.

Results

Over 45 weeks of testing, of 10,000 patient visits, 8,839 individual patient samples were available for analysis following removal of duplicates. A sustained target uptake of >50% was obtained after week 3. 97(1.09%), 44(0.49%) and 447(5.05%) HIV, Hepatitis B and Hepatitis C tests were positive respectively. Of these, 7(0.08%), 20(0.22%) and 58(0.66%) were new diagnoses of HIV, Hepatitis B and Hepatitis C respectively. The new diagnosis rate for HIV, Hepatitis B and Hepatitis C was 0.8, 2.26 and 6.5 per 1000 and study prevalence for HIV, Hepatitis B and Hepatitis C was 11.0, 5.0 and 50.5 per 1000 respectively.

Conclusions

Opt-out blood borne viral screening was feasible and acceptable in an inner-city ED. Blood borne viral infections were prevalent in this population and newly diagnosed cases were diagnosed and linked to care. These results suggest widespread blood borne viral testing in differing clinical locations with differing population demographic risks may be warranted.     

Characterization of Opioid Receptors in Cultured Neurons

The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors.     

Proline residues within spacer peptide p1 are important for human immunodeficiency virus type 1 infectivity,protein processing,and genomic RNA dimer stability

The full-length human immunodeficiency virus type 1 (HIV-1) mRNA encodes two precursor polyproteins, Gag and GagProPol. An infrequent ribosomal frameshifting event allows these proteins to be synthesized from the same mRNA in a predetermined ratio of 20 Gag proteins for each GagProPol. The RNA frameshift signal consists of a slippery sequence and a hairpin stem-loop whose thermodynamic stability has been shown in in vitro translation systems to be critical to frameshifting efficiency. In this study we examined the frameshift region of HIV-1, investigating the effects of altering stem-loop stability in the context of the complete viral genome and assessing the role of the Gag spacer peptide p1 and the GagProPol transframe (TF) protein that are encoded in this region. By creating a series of frameshift region mutants that systematically altered the stability of the frameshift stem-loop and the protein sequences of the p1 spacer peptide and TF protein, we have demonstrated the importance of stem-loop thermodynamic stability in frameshifting efficiency and viral infectivity. Multiple changes to the amino acid sequence of p1 resulted in altered protein processing, reduced genomic RNA dimer stability, and abolished viral infectivity. The role of the two highly conserved proline residues in p1 (position 7 and 13) was also investigated. Replacement of the two proline residues by leucines resulted in mutants with altered protein processing and reduced genomic RNA dimer stability that were also noninfectious. The unique ability of proline to confer conformational constraints on a peptide suggests that the correct folding of p1 may be important for viral function.