Calcium and pancreatic β-cell function 7. Evidence for cyclic AMP-induced translocation of intracellular calcium

The effect of cyclic AMP on calcium movements in the pancreatic β-cell was evaluated using an experimental approach based on in situ labelling of intracellular organelles of ob/ob-mouse islets with ⁴⁵Ca. Whereas the glucose-stimulated ⁴⁵Ca incorporation by mitochondria and secretory granules was increased under a condition known to reduce cyclic AMP (starvation), raised levels of this nucleotide (addition of 3-isobutyl-1-methylxanthine or N⁶,O^2′-dibutyryl adenosine 3′,5′-cyclic monophosphate) reduced the mitochondrial accumulation of ⁴⁵Ca. Conditions with increased cyclic AMP were associated with a stimulated efflux of ⁴⁵Ca from the secretory granules but not from the mitochondria. The microsomal fraction differed from both the mitochondrial and secretory granule fractions by accumulating more ⁴⁵Ca after the addition of 3-isobutyl-1-methylxanthine. The results suggest that cyclic AMP potentiates glucose-stimulated insulin release by increasing cytoplasmic Ca²⁺ at the expense of the calcium taken up by the organelles of the pancreatic β-cells.     

Identification of significant regions of transcription factor DP-1 (TFDP-1) involved in stability/instability of the protein

We previously reported the identification of DP-1 isoforms (α and β), which are structurally C-terminus-deleted ones, and revealed the low-level expression of these isoforms. It is known that wild-type DP-1 is degraded by the ubiquitin-proteasome system, but few details are known about the domains concerned with the protein stability/instability for the proteolysis of these DP-1 isoforms. Here we identified the domains responsible for the stability/instability of DP-1. Especially, the DP-1 “Stabilon” domain was a C-terminal acidic motif and was quite important for DP-1 stability. Moreover, we propose that this DP-1 Stabilon may be useful for the stability of other nuclear proteins when fused to them.     

Involvement of Kv1.3 and p38 MAPK signaling in HIV-1 glycoprotein 120-induced microglia neurotoxicity

Inflammatory responses mediated by activated microglia play a pivotal role in the pathogenesis of human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorders. Studies on identification of specific targets to control microglia activation and resultant neurotoxic activity are imperative. Increasing evidence indicate that voltage-gated K⁺ (K_v) channels are involved in the regulation of microglia functionality. In this study, we investigated K_v1.3 channels in the regulation of neurotoxic activity mediated by HIV-1 glycoprotein 120 (gp120)-stimulated rat microglia. Our results showed treatment of microglia with gp120 increased the expression levels of K_v1.3 mRNA and protein. In parallel, whole-cell patch-clamp studies revealed that gp120 enhanced microglia K_v1.3 current, which was blocked by margatoxin, a K_v1.3 blocker. The association of gp120 enhancement of K_v1.3 current with microglia neurotoxicity was demonstrated by experimental results that blocking microglia K_v1.3 attenuated gp120-associated microglia production of neurotoxins and neurotoxicity. Knockdown of K_v1.3 gene by transfection of microglia with K_v1.3-siRNA abrogated gp120-associated microglia neurotoxic activity. Further investigation unraveled an involvement of p38 MAPK in gp120 enhancement of microglia K_v1.3 expression and resultant neurotoxic activity. These results suggest not only a role K_v1.3 may have in gp120-associated microglia neurotoxic activity, but also a potential target for the development of therapeutic strategies.     

Predation as the primary selective force in recurrent evolution of gigantism in Poecilozonites land snails in Quaternary Bermuda

During the last half million years, pulses of gigantism in the anagenetic lineage of land snails of the subgenus Poecilozonites on Bermuda were correlated with glacial periods when lower sea level resulted in an island nearly an order of magnitude larger than at present. During those periods, the island was colonized by large vertebrate predators that created selection pressure for large size and rapid growth in the snails. Extreme reduction in land area from rising seas, along with changes in ecological conditions at the onset of interglacial episodes, marked extinction events for large predators, after which snails reverted to much smaller size. The giant snails were identical in morphology during the last two glacials when the predators included a large flightless rail Rallus recessus (marine isotope stages (MIS) 4-2) and a crane Grus latipes and a duck Anas pachysceles (MIS 6). In a preceding glacial period (MIS 10), when the fauna also included the tortoise Hesperotestudo bermudae, the snails were not only large, but the shells were much thicker, presumably to prevent crushing by tortoises. Evolution of Poecilozonites provides an outstanding example of dramatic morphological change in response to environmental pressures in the absence of cladogenesis.     

Ageing and thermal performance in the sub-Antarctic wingless fly Anatalanta aptera (Diptera: Sphaeroceridae): older is better

Senescence is a progressive biological process expressed in behavioural, morphological, physiological, biochemical and cellular age-related changes. Age-associated alterations in activity are regularly found in insects when examining whole-organism senescence over the adult lifespan. In addition, overall stress resistance usually decreases with senescence. In the present study, we measured the critical thermal minimum (CT_min) and the subsequent recovery period over the lifespan of the sub-Antarctic wingless fly, Anatalanta aptera. Experiments were conducted on males and females in seven age groups: newly emerged, 1.5-, 5-, 7-, 13-, 15- and 18-month-old adults. Surprisingly, CT_min decreased significantly with ageing in A. aptera, from −3.8 ± 0.5°C just after the emergence to −5.6 ± 0.7°C in the 18-month-old flies. The subsequent recovery period remained similar between the seven groups tested. Our unexpected results contradict the previous data collected in other insects. We have demonstrated for the first time that ageing may improve rather than impair locomotor activity during unfavourable thermal conditions. It raises questions and challenges the literature dealing with ageing. These fascinating results also question the underpinning mechanisms involved in the improvement of the thermal performance with ageing in A. aptera.     

TP53 PIN3 and PEX4 polymorphisms and infertility associated with endometriosis or with post-in vitro fertilization implantation failure

p53 has a crucial role in human fertility by regulating the expression of leukemia inhibitory factor (LIF), a secreted cytokine critical for blastocyst implantation. To examine whether TP53 polymorphisms may be involved with in vitro fertilization (IVF) failure and endometriosis (END), we have assessed the associations between TP53 polymorphism in intron 2 (PIN2; G/C, intron 2), PIN3 (one (N, non-duplicated) or two (D, duplicated) repeats of a 16-bp motif, intron 3) and polymorphism in exon 4 (PEX4; C/G, p.P72R, exon 4) in 98 women with END and 115 women with post-IVF failure. In addition, 134 fertile women and 300 women unselected with respect to fertility-related features were assessed. TP53 polymorphisms and haplotypes were identified by amplification refractory mutation system polymerase chain reaction. TP53 PIN3 and PEX4 were associated with both END (P=0.042 and P=0.007, respectively) and IVF (P=0.004 and P=0.009, respectively) when compared with women both selected and unselected for fertility-related features. Haplotypes D-C and N-C were related to higher risk for END (P=0.002, P=0.001, respectively) and failure of IVF (P=0.018 and P=0.002, respectively) when compared with the Fertile group. These results support that specific TP53 haplotypes are associated with an increased risk of END-associated infertility and with post-IVF failure.     

Genome-wide analysis of N1-methyl-adenosine modification in human tRNAs

The N¹-methyl-Adenosine (m¹A58) modification at the conserved nucleotide 58 in the TΨC loop is present in most eukaryotic tRNAs. In yeast, m¹A58 modification is essential for viability because it is required for the stability of the initiator-tRNA^Met. However, m¹A58 modification is not required for the stability of several other tRNAs in yeast. This differential m¹A58 response for different tRNA species raises the question of whether some tRNAs are hypomodified at A58 in normal cells, and how hypomodification at A58 may affect the stability and function of tRNA. Here, we apply a genomic approach to determine the presence of m¹A58 hypomodified tRNAs in human cell lines and show how A58 hypomodification affects stability and involvement of tRNAs in translation. Our microarray-based method detects the presence of m¹A58 hypomodified tRNA species on the basis of their permissiveness in primer extension. Among five human cell lines examined, approximately one-quarter of all tRNA species are hypomodified in varying amounts, and the pattern of the hypomodified tRNAs is quite similar. In all cases, no hypomodified initiator-tRNA^Met is detected, consistent with the requirement of this modification in stabilizing this tRNA in human cells. siRNA knockdown of either subunit of the m¹A58-methyltransferase results in a slow-growth phenotype, and a marked increase in the amount of m¹A58 hypomodified tRNAs. Most m¹A58 hypomodified tRNAs can associate with polysomes in varying extents. Our results show a distinct pattern for m¹A58 hypomodification in human tRNAs, and are consistent with the notion that this modification fine tunes tRNA functions in different contexts.     

Circadian communication between unicells?

Populations ofGonyaulax polyedra, in two different phases, about 11 h apart, were mixed, and the intensity of their spontaneous bioluminescence glow recorded for about 2 wk under conditions of constant dim (35±3 μE/m²/s) white light and constant temperature (19.0±0.3°C). The phases and amplitudes of glow signals recorded from mixed cultures were compared with those obtained from the arithmetic sum of the intensity data from two control vials. Peaks in control cultures generally remained separate, but there was a spontaneous increase in the period beginning 6–11 d after the onset of constant conditions. This did not occur in cultures in which the medium was exchanged with fresh medium every 2 d. In the actual mixes of two cultures there was a merging of the two subpeaks in the signal, which did not occur when the medium was exchanged. The results indicate that conditioning of the medium by cells may affect the period of the circadian rhythm and that this might result in a type of communication. Supported by the Deutsche Forschungsgemeinschaft; present address