Numb and alpha-Adaptin regulate Sanpodo endocytosis to specify cell fate in Drosophila external sensory organs

During asymmetric cell division in Drosophila sensory organ precursors (SOPs), the Numb protein segregates into one of the two daughter cells, in which it inhibits Notch signalling to specify pIIb cell fate. We show here that Numb acts in SOP cells by inducing the endocytosis of Sanpodo, a four-pass transmembrane protein that has previously been shown to regulate Notch signalling in the central nervous system. In sanpodo mutants, SOP cells divide symmetrically into two pIIb cells. We show that Sanpodo is cortical in pIIa, but colocalizes with Notch and Delta in Rab5- and Rab7-positive endocytic vesicles in pIIb. Sanpodo endocytosis requires alpha-Adaptin, a Numb-binding partner involved in clathrin-mediated endocytosis. In numb or alpha-adaptin mutants, Sanpodo is not endocytosed. Surprisingly, this defect is observed already before and during mitosis, which suggests that Numb not only acts in pIIb, but also regulates endocytosis throughout the cell cycle. Numb binds to Sanpodo by means of its phosphotyrosine-binding domain, a region that is essential for Numb function. Our results establish numb- and alpha-adaptin-dependent endocytosis of Sanpodo as the mechanism by which Notch is regulated during external sensory organ development.     

Iron and proteins for iron storage and detoxification

Iron is required by most organisms, but is potentially toxic due to the low solubility of the stable oxidation state, Fe(III), and to the tendency to potentiate the production of reactive oxygen species, ROS. The reactivity of iron is counteracted by bacteria with the same strategies employed by the host, namely by sequestering the metal into ferritin, the ubiquitous iron storage protein. Ferritins are highly conserved, hollow spheres constructed from 24 subunits that are endowed with ferroxidase activity and can harbour up to 4500 iron atoms as oxy-hydroxide micelles. The release of the metal upon reduction can alter the microorganism-host iron balance and hence permit bacteria to overcome iron limitation. In bacteria, the relevance of the Dps (DNA-binding proteins from starved cells) family in iron storage-detoxification has been recognized recently. The seminal studies on the protein from Listeria innocua demonstrated that Dps proteins have ferritin-like activity and most importantly have the capacity to attenuate the production of ROS. This latter function allows bacterial pathogens that lack catalase, e.g. Porphyromonas gingivalis, to survive in an aerobic environment and resist to peroxide stress.     

(-)-Amarbellisine, a lycorine-type alkaloid from Amaryllis belladonna L. growing in Egypt

A new lycorine-type alkaloid, named (-)-amarbellisine, was isolated from the bulbs of Egyptian Amaryllis belladonna L. together with the well known alkaloids (-)-lycorine, (-)-pancracine, (+)-vittatine, (+)-11-hydroxyvittatine, and (+)-hippeastrine. The new alkaloid, containing the pyrrolo[de]phenanthridine ring system, was essentially characterised by spectroscopic and optical methods, and proved to be the 2-methoxy-3a,4,5,7,11b,11c-hexahydro-1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridinol. By using HPTLC technique we also carried out a comparative study of the relative and total alkaloidal content at two different stages of plant growth. Finally, the antimicrobial activity of the isolated alkaloids was assayed.     

Identification and Quantification of a New Family of Peptide Endocannabinoids (Pepcans) Showing Negative Allosteric Modulation at CB1 Receptors

The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB₁). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB₁ receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [³H]CP55,940 and [³H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [³H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB₁ receptors. Competition binding studies revealed K_i values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and ∼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [³⁵S]GTPγS binding, and CB₁ receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB₁ receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling.     

Utilization of Sialylated Glycans as Coreceptors Enhances the Neurovirulence of Serotype 3 Reovirus

Mammalian reoviruses display serotype-specific patterns of tropism and disease in the murine central nervous system (CNS) attributable to polymorphisms in viral attachment protein σ1. While all reovirus serotypes use junctional adhesion molecule-A as a cellular receptor, they differ in their utilization of carbohydrate coreceptors. This observation raises the possibility that carbohydrate binding by σ1 influences reovirus pathology in the CNS. In this study, we sought to define the function of carbohydrate binding in reovirus neuropathogenesis. Newborn mice were inoculated intramuscularly with wild-type strain type 3 Dearing (T3D) and T3D-σ1R202W, a point mutant T3D derivative that does not bind sialic acid (SA). Infected mice were monitored for survival, and viral loads at the sites of primary and secondary replication were quantified. Fewer mice inoculated with the wild-type virus survived in comparison to those inoculated with the mutant virus. The wild-type virus also produced higher titers in the spinal cord and brain at late times postinoculation but lower titers in the liver in comparison to those produced by the mutant virus. In addition, the wild-type virus was more virulent and produced higher titers in the brain than the mutant following intracranial inoculation. These animal infectivity studies suggest that T3D-σ1R202W harbors a defect in neural growth. Concordantly, compared with the wild-type virus, the mutant virus displayed a decreased capacity to infect and replicate in primary cultures of cortical neurons, a property dependent on cell surface SA. These results suggest that SA binding enhances the kinetics of reovirus replication in neural tissues and highlight a functional role for sialylated glycans as reovirus coreceptors in the CNS.     

Modulation of urinary peptidome in humans exposed to high altitude hypoxia

The exposure of healthy subjects to high altitude represents a model to explore the pathophysiology of diseases related to tissue hypoxia and to evaluate pharmacological approaches potentially useful as a therapy for chronic diseases related to hypoxia. We explored the urinary peptidome to detect alterations induced by the exposure of subjects to different altitudes (sea level, high altitude = 3500 m, very high altitude = 5400 m) and to pharmacological treatment. Urine samples were collected from 47 subjects, randomly and blindly assigned to placebo (n = 24) or Telmisartan (n = 23). Samples were purified by the use of magnetic beads, then analysed by MALDI-TOF MS. Results showed that the urinary peptidome is not affected by the administration of Telmisartan, neither at the sea level nor at high and very high altitudes. In contrast, the urinary protein profiles are modified when subjects are exposed to high and very high altitudes, and we detected six peptides differentially expressed in hypobaric hypoxia at high or very high altitude compared to the sea level. Two of them were identified as fragments of the glycoprotein uromodulin and of the α1-antitrypsin. This is the first proteomic study showing that hypobaric hypoxia conditions affect the urinary peptidome.     

Low temperature tolerance and starvation ability of the oak processionary moth: implications in a context of increasing epidemics

• 1 We investigated how modifications in winter and spring temperature conditions may affect the survival of a spring‐hatching Lepidoptera, the oak processionary moth Thaumetopoea processionea.
• 2 Supercooling and chilling injury experiments indicate that eggs are especially cold hardy at the start of the winter period, although this ability is reduced later in the season. In the spring, young larvae are sufficiently cold hardy to ensure no direct mortality as a result of late frosts.
• 3 A comparison of phenological models shows that neonate larvae may await the unfolding of new oak leaves for relatively long periods (e.g. 1–30 days). Under both low (4°C after 5 days at 16°C) and high temperature experimental scenarios (constant 16°C), the majority of neonate larvae can survive starvation for more than 2 weeks.
• 4 Larvae may also suffer from food depletion once their development has been initiated (e.g. during cold springs) if the threshold temperature for feeding is not reached for several consecutive days, or in the case of late frosts affecting foliage availability. When temperature is reduced to 4°C, developing larvae become inactive and do not feed anymore; their starvation survival capability is reduced to approximately 2 weeks (cold spring hypothesis). At 16°C, developing larvae that are deprived of food can only survive for 10 days (late frost hypothesis).
• 5 We conclude that, in the oak processionary moth, neonate larvae are relatively well adapted to early hatching relative to budburst, ensuring them the highest foliage quality for development. In some years, however, phenological asynchrony or cold spring conditions may affect the persistence of populations at the limits of the species' range.