Changes in plasma membrane Ca-ATPase and stromal interacting molecule 1 expression levels for Ca2+ signaling in dystrophic mdx mouse muscle

The majority of the skeletal muscle plasma membrane is internalized as part of the tubular (t-) system, forming a standing junction with the sarcoplasmic reticulum (SR) membrane throughout the muscle fiber. This arrangement facilitates not only a rapid and large release of Ca(2+) from the SR for contraction upon excitation of the fiber, but has also direct implications for other interdependent cellular regulators of Ca(2+). The t-system plasma membrane Ca-ATPase (PMCA) and store-operated Ca(2+) entry (SOCE) can also be activated upon release of SR Ca(2+). In muscle, the SR Ca(2+) sensor responsible for rapidly activated SOCE appears to be the stromal interacting molecule 1L (STIM1L) isoform of STIM1 protein, which directly interacts with the Orai1 Ca(2+) channel in the t-system. The common isoform of STIM1 is STIM1S, and it has been shown that STIM1 together with Orai1 in a complex with the partner protein of STIM (POST) reduces the activity of the PMCA. We have previously shown that Orai1 and STIM1 are upregulated in dystrophic mdx mouse muscle, and here we show that STIM1L and PMCA are also upregulated in mdx muscle. Moreover, we show that the ratios of STIM1L to STIM1S in wild-type (WT) and mdx muscle are not different. We also show a greater store-dependent Ca(2+) influx in mdx compared with WT muscle for similar levels of SR Ca(2+) release while normal activation and deactivation properties were maintained. Interestingly, the fiber-averaged ability of WT and mdx muscle to extrude Ca(2+) via PMCA was found to be the same despite differences in PMCA densities. This suggests that there is a close relationship among PMCA, STIM1L, STIM1S, Orai1, and also POST expression in mdx muscle to maintain the same Ca(2+) extrusion properties as in the WT muscle.     

Effect of creatine on contractile force and sensitivity in mechanically skinned single fibers from rat skeletal muscle

Increasing the intramuscular stores of total creatine [TCr = creatine (Cr) + creatine phosphate (CrP)] can result in improved muscle performance during certain types of exercise in humans. Initial uptake of Cr is accompanied by an increase in cellular water to maintain osmotic balance, resulting in a decrease in myoplasmic ionic strength. Mechanically skinned single fibers from rat soleus (SOL) and extensor digitorum longus (EDL) muscles were used to examine the direct effects on the contractile apparatus of increasing [Cr], increasing [Cr] plus decreasing ionic strength, and increasing [Cr] and [CrP] with no change in ionic strength. Increasing [Cr] from 19 to 32 mM, accompanied by appropriate increases in water to maintain osmolality, had appreciable beneficial effects on contractile apparatus performance. Compared with control conditions, both SOL and EDL fibers showed increases in Ca²⁺ sensitivity (+0.061 ± 0.004 and +0.049 ± 0.009 pCa units, respectively) and maximum Ca²⁺-activated force (to 104 ± 1 and 105 ± 1%, respectively). In contrast, increasing [Cr] alone had a small inhibitory effect. When both [Cr] and [CrP] were increased, there was virtually no change in Ca²⁺ sensitivity of the contractile apparatus, and maximum Ca²⁺-activated force was 106 ± 1% compared with control conditions in both SOL and EDL fibers. These results suggest that the initial improvement in performance observed with Cr supplementation is likely due in large part to direct effects of the accompanying decrease in myoplasmic ionic strength on the properties of the contractile apparatus. ergogenic aid; muscle contraction; fatigue     

A gain-of-function mutation in the second tetratricopeptide repeat of TFIIIC131 relieves autoinhibition of Brf1 binding

The interaction between the tetratricopeptide repeat (TPR)-containing subunit of TFIIIC, TFIIIC131, and the TFIIB-related factor Brf1 represents a limiting step in the assembly of the RNA polymerase III (pol III) initiation factor TFIIIB. This assembly reaction is facilitated by dominant mutations that map in and around TPR2. Structural modeling of TPR1 to TPR3 from TFIIIC131 shows that one such mutation, PCF1-2, alters a residue in the ligand-binding groove of the TPR superhelix whereas another mutation, PCF1-1, changes a surface-accessible residue on the back side of the TPR superhelix. In this work, we show that the PCF1-1 mutation (H190Y) increases the binding affinity for Brf1, but does not affect the binding affinity for Bdp1, in the TFIIIC-dependent assembly of TFIIIB. Interestingly, binding studies with TFIIIC131 fragments indicate that Brf1 does not interact directly at the site of the PCF1-1 mutation. Rather, the data suggest that the mutation overcomes the previously documented autoinhibition of Brf1 binding. These findings together with the results from site-directed mutagenesis support the hypothesis that gain-of-function mutations at amino acid 190 in TPR2 stabilize an alternative conformation of TFIIIC131 that promotes its interaction with Brf1.     

Na+-dependent transport of large neutral amino acids occurs at the abluminal membrane of the blood-brain barrier

Several Na+-dependent carriers of amino acids exist on the abluminal membrane of the blood-brain barrier (BBB). These Na+-dependent carriers are in a position to transfer amino acids from the extracellular fluid of brain to the endothelial cells and thence to the circulation. To date, carriers have been found that may remove nonessential, nitrogen-rich, or acidic (excitatory) amino acids, all of which may be detrimental to brain function. We describe here Na+-dependent transport of large neutral amino acids across the abluminal membrane of the BBB that cannot be ascribed to currently known systems. Fresh brains, from cows killed for food, were used. Microvessels were isolated, and contaminating fragments of basement membranes, astrocyte fragments, and pericytes were removed. Abluminal-enriched membrane fractions from these microvessels were prepared. Transport was Na+ dependent, voltage sensitive, and inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a particular inhibitor of the facilitative large neutral amino acid transporter 1 (LAT1) system. The carrier has a high affinity for leucine (Km 21 +/- 7 microM) and is inhibited by other neutral amino acids, including glutamine, histidine, methionine, phenylalanine, serine, threonine, tryptophan, and tyrosine. Other established neutral amino acids may enter the brain by way of LAT1-type facilitative transport. The presence of a Na+-dependent carrier on the abluminal membrane capable of removing large neutral amino acids, most of which are essential, from brain indicates a more complex situation that has implications for the control of essential amino acid content of brain.     

The taxonomic treatment of agamosperms in the genusLimonium mill. (Plumbaginaceae)

Agamospermous species account for a large proportion of the species ofLimonium. Agamospermy is indicated by uneven polyploidy or aneuploidy, low pollen stainability and by the presence of monomorphic self-incompatible populations. The taxonomic treatment of agamospermous taxa varies from recognition of all of them at the same specific rank or by utilising a range of ranks in the taxonomic hierarchy. The influence of evolutionary hypotheses on taxonomic systems is considered. Molecular data provide a means of measuring the genetical relationships of taxa and establishing groups in a taxonomic hierarchy.     

Will vaccinated women attend cervical screening? A population based survey of human papillomavirus vaccination and cervical screening among young women in Victoria,Australia

Objectives: To assess human papillomavirus (HPV) vaccination coverage and attitudes to vaccination and Pap screening in young women. Design: Population-based telephone survey. Setting: Victoria, Australia. Participants: 234 women resident in Victoria aged 18–28 years in May 2009. Main outcome measures: Self-reported HPV vaccination uptake, reasons for non-receipt or failure to complete vaccination, knowledge and attitudes about HPV vaccination and Pap screening, and cervical screening intentions. Results: The response rate for eligible households was 62.4%. Half of the women (56%, n = 131) had previously had a Pap test and 74% (age standardised estimate) had received HPV vaccine. Of the vaccinated women, 5% had received one dose only, 18% two doses and 76% had completed the course (1.7% unsure of number of doses). Vaccination uptake was highest in the youngest women (declining from 90% for at least one dose in women aged 18–38.5% in women aged 28; p for trend <0.001). Among women who had heard of the vaccine, 96% knew Pap tests were still needed after it, although 20% thought the vaccine could prevent all cervical cancers and 9% thought the vaccine could treat cervical abnormalities and cancer. Among vaccinated women, 8% of women agreed that having been vaccinated made them less likely to have Pap tests in the future. Conclusions: Self-reported coverage in this sample was higher than that recorded on the national vaccination register. Young women report the message that Pap tests are required after vaccination, but there are gaps in their knowledge about the limitations of the vaccine so it remains to be seen if they actually follow through with having Pap tests. Ongoing monitoring of cervical screening rates will be important as this cohort ages.     

Selective Brain Cooling Reduces Water Turnover in Dehydrated Sheep

In artiodactyls, arterial blood destined for the brain can be cooled through counter-current heat exchange within the cavernous sinus via a process called selective brain cooling. We test the hypothesis that selective brain cooling, which results in lowered hypothalamic temperature, contributes to water conservation in sheep. Nine Dorper sheep, instrumented to provide measurements of carotid blood and brain temperature, were dosed with deuterium oxide (D₂O), exposed to heat for 8 days (40◦C for 6-h per day) and deprived of water for the last five days (days 3 to 8). Plasma osmolality increased and the body water fraction decreased over the five days of water deprivation, with the sheep losing 16.7% of their body mass. Following water deprivation, both the mean 24h carotid blood temperature and the mean 24h brain temperature increased, but carotid blood temperature increased more than did brain temperature resulting in increased selective brain cooling. There was considerable inter-individual variation in the degree to which individual sheep used selective brain cooling. In general, sheep spent more time using selective brain cooling, and it was of greater magnitude, when dehydrated compared to when they were euhydrated. We found a significant positive correlation between selective brain cooling magnitude and osmolality (an index of hydration state). Both the magnitude of selective brain cooling and the proportion of time that sheep spent selective brain cooling were negatively correlated with water turnover. Sheep that used selective brain cooling more frequently, and with greater magnitude, lost less water than did conspecifics using selective brain cooling less efficiently. Our results show that a 50kg sheep can save 2.6L of water per day (~60% of daily water intake) when it employs selective brain cooling for 50% of the day during heat exposure. We conclude that selective brain cooling has a water conservation function in artiodactyls.     

Very Few Substitutions in a Germ Line Antibody Are Required To Initiate Significant Domain Exchange

2G12 is a broadly neutralizing anti-HIV-1 monoclonal human IgG1 antibody reactive with a high-mannose glycan cluster on the surface of glycoprotein gp120. A key feature of this very highly mutated antibody is domain exchange of the heavy-chain variable region (V_H) with the V_H of the adjacent Fab of the same immunoglobulin, which assembles a multivalent binding interface composed of two primary binding sites in close proximity. A non-germ line-encoded proline in the elbow between V_H and C_H1 and an extensive network of hydrophobic interactions in the V_H/V_H′ interface have been proposed to be crucial for domain exchange. To investigate the origins of domain exchange, a germ line version of 2G12 that behaves as a conventional antibody was engineered. Substitution of 5 to 7 residues for those of the wild type produced a significant fraction of domain-exchanged molecules, with no evidence of equilibrium between domain-exchanged and conventional forms. Two substitutions not previously implicated, A^H14 and E^H75, are the most crucial for domain exchange, together with I^H19 at the V_H/V_H′ interface and P^H113 in the elbow region. Structural modeling gave clues as to why these residues are essential for domain exchange. The demonstration that domain exchange can be initiated by a small number of substitutions in a germ line antibody suggests that the evolution of a domain-exchanged antibody response in vivo may be more readily achieved than considered to date.Protein oligomers are able to exchange or swap an element of their secondary structure or an entire protein domain. The functional unit in domain-exchanged proteins thereby stays preserved, as only the linking hinge loop changes conformation significantly (4, 17, 27). Analogous to other domain-swapped proteins, antibodies can exchange an entire domain, in this case the heavy-chain variable region (V_H), with an equivalent heavy-chain variable region of an adjacent Fab (V_H′) within the same immunoglobulin (Ig) molecule (11). The advantages of domain-exchanged proteins, including antibodies, are higher local concentrations of active sites, a larger binding surface, and a potential secondary active site at the new subunit interface (27, 45). The one and only antibody shown to be domain exchanged to date is 2G12 (7, 11), but this arrangement is potentially possible for any Ig and could have been overlooked at least in some instances.2G12 is one of only a few high-affinity monoclonal antibodies with broad neutralizing activity against different subtypes of HIV-1 (5, 30, 40, 43). The antibody binds a dense cluster of N-linked high-mannose glycans (Man_8-9GlcNAc₂) on the envelope surface glycoprotein gp120 (10, 35, 36, 41). The domain-exchanged arrangement forms a multivalent binding site composed of two primary binding sites in close proximity and a proposed secondary binding site formed by the novel V_H/V_H′ interface (11). 2G12 provides protection against infection in animal models (19, 31) and has been shown to induce neutralization escape following passive immunization in humans (39).Consensus has grown that a successful HIV-1 vaccine will need to include a component that elicits broadly neutralizing antibodies (8, 18, 21, 26, 32, 42). All attempts to elicit 2G12-like antibodies with the desired specificity and neutralization activity have failed to date (22, 29, 44), conceivably due to difficulties in generating adequate mimicry of the glycan cluster and tolerance mechanisms or, very likely, the inability to induce domain exchange (1). Unraveling the mechanism of domain exchange and how this conformation might have evolved is highly desirable to direct future HIV-1 vaccine design to elicit 2G12-like antibodies.By comparison with other domain-exchanged proteins (27), the following three mechanisms have been proposed to contribute to the unique structure of 2G12 compared to the structure of a conventional antibody: destabilization of the “closed” V_H/V_L interface, conformational change in the elbow between V_H and C_H1, and an energetically favorable “open” V_H/V_H′ interface (11). Key residues involved in promoting domain exchange were predicted based on examination of interacting residues at the two interfaces and by the effects of alanine substitutions on the binding of wild-type 2G12 to gp120. However, the importance of these key residues for domain exchange was not directly demonstrated experimentally (11).Here, we explored the minimal requirements for domain exchange of 2G12, starting with a germ line version of the antibody that adopts a conventional antibody structure. Although wild-type 2G12 is heavily somatically mutated, only five to seven substitutions in the germ line version of the antibody were shown to produce a significant fraction of domain-exchanged molecules. The results suggest the evolution of domain-exchanged antibody responses may be more facile than considered to date.