Pom33, a novel transmembrane nucleoporin required for proper nuclear pore complex distribution

The biogenesis of nuclear pore complexes (NPCs) represents a paradigm for the assembly of high-complexity macromolecular structures. So far, only three integral pore membrane proteins are known to function redundantly in NPC anchoring within the nuclear envelope. Here, we describe the identification and functional characterization of Pom33, a novel transmembrane protein dynamically associated with budding yeast NPCs. Pom33 becomes critical for yeast viability in the absence of a functional Nup84 complex or Ndc1 interaction network, which are two core NPC subcomplexes, and associates with the reticulon Rtn1. Moreover, POM33 loss of function impairs NPC distribution, a readout for a subset of genes required for pore biogenesis, including members of the Nup84 complex and RTN1. Consistently, we show that Pom33 is required for normal NPC density in the daughter nucleus and for proper NPC biogenesis and/or stability in the absence of Nup170. We hypothesize that, by modifying or stabilizing the nuclear envelope–NPC interface, Pom33 may contribute to proper distribution and/or efficient assembly of nuclear pores.     

Vertebral body innervation: Implications for pain

Vertebral fractures often cause intractable pain. To define the involvement of vertebral body innervation in pain, we collected specimens from male and female patients during percutaneous kyphoplasty, a procedure used for reconstruction of the vertebral body. Specimens were taken from 31 patients (9 men and 22 women) suffering high‐intensity pain before surgery. In total, 1,876 histological preparations were obtained and analysed. Immunohistochemical techniques were used to locate the nerves in the specimens. The nerve fibres were labelled by indirect immunofluorescence with the primary antibody directed against Protein Gene Product 9.5 (PGP 9.5), a pan‐neuronal marker; another primary antibody directed against type IV collagen (Col IV) was used to identify vessels and to determine their relationship with vertebral nerve fibres. The mean percentage of samples in which it was possible to identify nerve fibres was 35% in men and 29% in women. The percentages varied depending on the spinal level considered and the sex of the subject, nerve fibres being mostly present around vessels (95%). In conclusion, there is scarce innervation of the vertebral bodies, with a clear prevalence of fibres located around vessels. It seems unlikely that this pattern of vertebral body innervation is involved in vertebral pain or in pain relief following kyphoplasty. J. Cell. Physiol. 222: 488–491, 2010. © 2009 Wiley‐Liss, Inc.     

Non-apoptotic programmed cell death induced by a copper(II) complex in human fibrosarcoma cells

A₀, a Cu(II) thioxotriazole complex, produces severe cytotoxic effects on HT1080 human fibrosarcoma cells with a potency comparable to that exhibited by cisplatin. A₀ induced a characteristic series of changes, hallmarked by the formation of eosin- and Sudan Black-B-negative vacuoles. No evidence of nuclear fragmentation or caspase-3 activation was detected in cells treated with A₀ which, rather, inhibited cisplatin-stimulated caspase-3 activity. Membrane functional integrity, assessed with calcein and propidium iodide, was spared until the late stages of the death process induced by the copper complex. Vacuoles were negative to the autophagy marker monodansylcadaverine and their formation was not blocked by 3-methyladenine, an inhibitor of autophagic processes. Negativity to the extracellular marker pyranine excluded vacuole derivation from the extracellular fluid. Ultrastructural analysis indicated that A₀ caused the appearance of many electronlight cytoplasmic vesicles, possibly related to the endoplasmic reticulum, which progressively enlarge and coalesce to form large vacuolar structures that eventually fill the cytoplasm. It is concluded that A₀ triggers a non-apoptotic, type 3B programmed cell death (Clarke in Anat Embryol (Berl) 181:195–213, 1990), characterized by an extensive cytoplasmic vacuolization. This peculiar cytotoxicity pattern may render the employment of A₀ to be of particular interest in apoptosis-resistant cell models.     

Genetic identification of neurons controlling a sexually dimorphic behaviour

In the fruit fly Drosophila melanogaster, locomotor activity is sexually dimorphic: female flies constantly modulate their activity pattern whereas males show a steadier, stereotyped walking pace [1]. Here, we mapped the area of the brain controlling this behavioural dimorphism. Adult male Drosophila expressing a dominant feminising transgene in a small cluster of neurons in the pars intercerebralis exhibited a female-like pattern of locomotor activity. Genetic ablation of these neurons prevented the feminisation of the locomotor activity of transgenic males. The results suggest that this cluster of neurons modulates sex-specific activity, but is not involved in initiating fly locomotion. Nor does it control male courtship behaviour, because feminisation of courtship was not correlated with the feminisation of locomotor activity.     

Common copy number variations in fifty radiosensitive cell lines

Hypersensitivity to radiation exposure is a major challenge to radiotherapy in the treatment of cancer patients. Copy number variations (CNVs) are believed to identify genomic regions of functional significance for radiosensitivity (RS) but have yet to be systematically investigated. We used Affymetrix 6.0 SNP arrays to survey common CNVs in a cohort of 50 radiosensitive lymphoblastoid cell lines (RS-LCLs) derived from patients with undiagnosed diseases. A total of 317 CNVs that were present in at least 10% of the studied cell lines were identified. Three hundred and eight CNVs overlapped with polymorphic CNVs, 13 of which were significantly enriched in the RS-LCLs compared to the reference. The remaining 9 CNVs were novel. The majority of these enriched and novel CNVs were chromosomal gains. The dominance of the chromosomal gains over losses is inconsistent with the traditional concept of molecular basis of RS and suggests more complex genetic mechanisms for RS.     

A rapid method to increase the number of F(1) plants in pea (Pisum sativum) breeding programs

In breeding programs, a large number of F(2) individuals are required to perform the selection process properly, but often few such plants are available. In order to obtain more F(2) seeds, it is necessary to multiply the F(1) plants. We developed a rapid, efficient and reproducible protocol for in vitro shoot regeneration and rooting of seeds using 6-benzylaminopurine. To optimize shoot regeneration, basic medium contained Murashige and Skoog (MS) salts with or without B5 Gamborg vitamins and different concentrations of 6-benzylaminopurine (25, 50 and 75 μM) using five genotypes. We found that modified MS (B5 vitamins + 25 μM 6-benzylaminopurine) is suitable for in vitro shoot regeneration of pea. Thirty-eight hybrid combinations were transferred onto selected medium to produce shoots that were used for root induction on MS medium supplemented with α-naphthalene-acetic acid. Elongated shoots were developed from all hybrid genotypes. This procedure can be used in pea breeding programs and will allow working with a large number of plants even when the F(1) plants produce few seeds.     

A new 2CTAB/PCI method improves DNA amplification success from faeces of Mediterranean (Barbary macaques) and tropical (lowland gorillas) primates

Animal genomic DNA extracts of sufficient quality to address questions about population biology or behavioural ecology can be obtained from faeces when adequate extraction procedures are used. The presence of PCR inhibitors in extract products appears generally the main factor limiting DNA amplification success. We compared DNA amplification success from faeces of a tropical primate (western lowland gorilla, Gorilla g. gorilla) and a Mediterranean primate (Barbary macaque, Macaca sylvanus) between a standardized extraction technique widely used in animals (QIAamp^® stool kit), a technique mainly used in plant species (CTAB) and a new protocol (2CTAB/PCI). Amplification success varied from 51% to 97%, the highest success being reached with the 2CTAB/PCI protocol in both species.