Reduced levels of Cacna1c attenuate mesolimbic dopamine system function

Genetic variation in CACNA1C, which codes for the L‐type calcium channel (LTCC) Ca_v1.2, is associated with clinical diagnoses of bipolar disorder, depression and schizophrenia. Dysregulation of the mesolimbic‐dopamine (ML‐DA) system is linked to these syndromes and LTCCs are required for normal DAergic neurotransmission between the ventral tegmental area (VTA) and nucleus accumbens (NAc). It is unclear, however, how variations in CACNA1C genotype, and potential subsequent changes in expression levels in these regions, modify risk. Using constitutive and conditional knockout mice, and treatment with the LTCC antagonist nimodipine, we examined the role of Cacna1c in DA‐mediated behaviors elicited by psychomotor stimulants. Using fast‐scan cyclic voltammetry, DA release and reuptake in the NAc were measured. We find that subsecond DA release in Cacna1c haploinsufficient mice lacks normal sensitivity to inhibition of the DA transporter (DAT). Constitutive haploinsufficiency of Cacna1c led to attenuation of hyperlocomotion following acute administration of stimulants specific to DAT, and locomotor sensitization of these mice to the DAT antagonist GBR12909 did not reach the same level as wild‐type mice. The maintenance of sensitization to GBR12909 was attenuated by administration of nimodipine. Sensitization to GBR12909 was attenuated in mice with reduced Cacna1c selectively in the VTA but not in the NAc. Our findings show that Cacna1c is crucial for normal behavioral responses to DA stimulants and that its activity in the VTA is required for behavioral sensitization. Cacna1c likely exerts these effects through modifications to presynaptic ML‐DA system function.     

Multi-Serotype Pneumococcal Nasopharyngeal Carriage Prevalence in Vaccine Na?ve Nepalese Children,Assessed Using Molecular Serotyping

Invasive pneumococcal disease is one of the major causes of death in young children in resource poor countries. Nasopharyngeal carriage studies provide insight into the local prevalence of circulating pneumococcal serotypes. There are very few data on the concurrent carriage of multiple pneumococcal serotypes. This study aimed to identify the prevalence and serotype distribution of pneumococci carried in the nasopharynx of young healthy Nepalese children prior to the introduction of a pneumococcal conjugate vaccine using a microarray-based molecular serotyping method capable of detecting multi-serotype carriage. We conducted a cross-sectional study of healthy children aged 6 weeks to 24 months from the Kathmandu Valley, Nepal between May and October 2012. Nasopharyngeal swabs were frozen and subsequently plated on selective culture media. DNA extracts of plate sweeps of pneumococcal colonies from these cultures were analysed using a molecular serotyping microarray capable of detecting relative abundance of multiple pneumococcal serotypes. 600 children were enrolled into the study: 199 aged 6 weeks to <6 months, 202 aged 6 months to < 12 months, and 199 aged 12 month to 24 months. Typeable pneumococci were identified in 297/600 (49·5%) of samples with more than one serotype being found in 67/297 (20·2%) of these samples. The serotypes covered by the thirteen-valent pneumococcal conjugate vaccine were identified in 44·4% of samples containing typeable pneumococci. Application of a molecular serotyping approach to identification of multiple pneumococcal carriage demonstrates a substantial prevalence of co-colonisation. Continued surveillance utilising this approach following the introduction of routine use of pneumococcal conjugate vaccinates in infants will provide a more accurate understanding of vaccine efficacy against carriage and a better understanding of the dynamics of subsequent serotype and genotype replacement.     

The social development of free-ranging infantLemur catta at Berenty reserve,Madagascar

The social development of 11 free-ranging infantLemur catta was examined over the first 16 weeks of the infants' lives. By 16 weeks, infants still occasionally suckled and were carried dorsally, but on the whole, they were independent of their mothers. Sex or mother's rank was not found to affect frequency or type of play behavior. Mother's rank had no effect on frequency of maternal rejections, from the nipple or from riding, but female infants were rejected slightly more frequently than males were. Mothers tended to reject infants more severely and more frequently from dorsal riding than from the nipple. Sex and rank differences were not found with respect to behaviors determined as measures of independence; however, lower-ranking infants engaged in significantly more dependent behaviors than higher-ranking young did. It may be necessary for the infant of a low-ranking mother to maintain closer proximity to its mother for a longer period of time during infancy because such infants may be subject to abuse by higher-ranking group members and, furthermore, may not be as readily rescued in a stressful or dangerous situation as a higher-ranking infant. Sex was not found to be a factor in terms of measures of dependence. The lack of sex differences in developmental behaviors in this species may be related to female dominance, as well as to the fact that, as adults, both sexes engage in aggressive territorial behavirs.     

Arfophilins are dual Arf/Rab 11 binding proteins that regulate recycling endosome distribution and are related to Drosophila nuclear fallout

Arfophilin is an ADP ribosylation factor (Arf) binding protein of unknown function. It is identical to the Rab11 binding protein eferin/Rab11-FIP3, and we show it binds both Arf5 and Rab11. We describe a related protein, arfophilin-2, that interacts with Arf5 in a nucleotide-dependent manner, but not Arf1, 4, or 6 and also binds Rab11. Arfophilin-2 localized to a perinuclear compartment, the centrosomal area, and focal adhesions. The localization of arfophilin-2 to the perinuclear compartment was selectively blocked by overexpression of Arf5-T31N. In contrast, a green fluorescent protein-arfophilin-2 chimera or arfophilin-2 deletions were localized around the centrosome in a region that was also enriched for transferrin receptors and Rab11 but not early endosome markers, suggesting that the distribution of the endosomal recycling compartment was altered. The arfophilins belong to a conserved family that includes Drosophila melanogaster nuclear fallout, a centrosomal protein required for cellularization. Expression of green fluorescent protein-nuclear fallout in HeLa cells resulted in a similar phenotype, indicative of functional homology and thus implicating the arfophilins in mitosis/cytokinesis. We suggest that the novel dual GTPase-binding capacity of the arfophilins could serve as an interface of signals from Rab and Arf GTPases to regulate membrane traffic and integrate distinct signals in the late endosomal recycling compartment.     

Basis for Phospholipid Incorporation into Peripheral Nerve Myelin

Abstract: To characterize the mechanism(s) for targeting of phospholipids to peripheral nerve myelin, we examined the kinetics of incorporation of tritiated choline-, glycerol-, and ethanolamine-labeled phospholipids into four subfractions: microsomes, mitochondria, myelin-like material, and purified myelin at 1, 6, and 24 h after precursors were injected into sciatic nerves of 23–24-day-old rats. As validation of the fractionation scheme, a lag (> 1 h) in the accumulation of labeled phospholipids in the myelin-containing subfractions was found. This lag signifies the time between synthesis on organelles in Schwann cell cytoplasm and transport to myelin. In the present study, we find that sphingomyelin (choline-labeled) accumulated in myelin-rich subfractions only at 6 and 24 h, whereas phosphatidylserine (glycerol-labeled) and plasmalogen (ethanolamine-labeled) accumulated in the myelin-rich fractions by 1 h. The later phospholipids accumulate preferentially in the myelin-like fraction. These results are consistent with the notion that the targeting of sphingomyelin, a lipid present in the outer myelin leaflet, is different from the targeting of phosphatidylserine and ethanolamine plasmalogen, lipids in the inner leaflet. These findings are discussed in light of the possibility that sphingomyelin targeting is Golgi apparatus based, whereas phosphatidylserine and ethanolamine plasmalogen use a more direct transport system. Furthermore, the routes of phospholipid targeting mimic routes taken by myelin proteins P₀ (Golgi) and myelin basic proteins (more direct).     

DNA repair and replication in human fibroblasts treated with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

DNA repair and replication were examined in diploid human fibroblasts after treatment with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Unscheduled DNA synthesis exhibited a linear response to BPDE-I concentrations up to 1.5 μM and a saturation plateau after higher concentrations. Maximal unscheduled DNA synthesis was observed in the first hour after treatment with synthesis diminishing progressively thereafter. Half-maximal unscheduled DNA synthesis was seen within 4–6 h after treatment with 0.7 μM BPDE-I. DNA replication was inhibited by BPDE-I in a dose- and time-dependent fashion. The mechanisms of this inhibition were characterized by velocity sedimentation of pulse-labeled nascent DNA in alkaline sucrose gradients. Very low concentrations of BPDE-I (0.03 and 0.07 μM) were found to inhibit replicon initiation by up to 50% within 30–60 min after treatment. Recovery of initiation following these low concentrations was evident within 3 h after treatment. Higher concentrations of carcinogen inhibited DNA synthesis in active replicons. This effect was manifested by a reduction in incorporation of precursor into replication intermediates of greater than 1·10⁷ Da with the concurrent production of abnormally small nascent DNA. When viewed 45 min after treatment with 0.17 μM BPDE-I the combination of these two effects partially masked the inhibition of replicon initiation. However, even after treatment with 0.33 μM BPDE-I an effect on initiation was evident. These results reveal a pattern of response to BPDE-I that is quite similar to that produced by 254 nm radiation.     

Residency Duration and Shelter Quality Influence Vibratory Signalling Displays in A Territorial Caterpillar

Residents are more likely to win territorial disputes than intruders. One explanation for this prior resident advantage is that residents place a higher value on the resource and are therefore more motivated to win. Although value asymmetry models of animal contests often assume that contestants use information about resource value, information on the proximate cues affecting territorial behaviour is often lacking. We use a simple model system – territorial behaviour in the masked birch caterpillar (Drepana arcuata) to identify factors that affect territorial behaviour. Late instar caterpillars occupy solitary silken leaf shelters, which they defend against wandering conspecifics with a vibratory display. We evaluated how a caterpillar identifies itself as the owner and the factors that influence a resident's motivation to signal. To do so, we conducted three experiments between size‐matched residents and intruders to assess how residency duration and shelter quality independently affected territorial displays during the early stages of a contest. Experiment 1 (Time Exp.) demonstrated that resident signalling rates increase with increased duration on the leaf prior to introducing the intruder. Residents also signal more than intruders after residency periods of 1–3 min and longer, demonstrating that residents gather information about resource value shortly after occupying a leaf. Experiment 2 (Squatter Exp.) aimed to disentangle the effects of time on the leaf and silk accumulation. Squatters (individuals in a shelter made by another) placed for 1–3 min on a leaf containing a full silk shelter signalled more to intruders than did caterpillars placed on a fresh leaf for 1–3 min. Experiment 3 (Shelter Removal Exp.) showed that residents whose shelters had been removed signal less than those occupying an intact shelter, despite an equal length of time investing in them. Our experiment is the first to covary both prior residency duration and territory quality, and we find that the motivation of caterpillars to signal is a function of both of these attributes.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.