Engineered viruses to select genes encoding secreted and membrane-bound proteins in mammalian cells

We have developed a functional genomics tool to identify the subset of cDNAs encoding secreted and membrane-bound proteins within a library (the ‘secretome’). A Sindbis virus replicon was engineered such that the envelope protein precursor no longer enters the secretory pathway. cDNA fragments were fused to the mutant precursor and expression screened for their ability to restore membrane localization of envelope proteins. In this way, recombinant replicons were released within infectious viral particles only if the cDNA fragment they contain encodes a secretory signal. By using engineered viral replicons to selectively export cDNAs of interest in the culture medium, the methodology reported here efficiently filters genetic information in mammalian cells without the need to select individual clones. This adaptation of the ‘signal trap’ strategy is highly sensitive (1/200 000) and efficient. Indeed, of the 2546 inserts that were retrieved after screening various libraries, more than 97% contained a putative signal peptide. These 2473 clones encoded 419 unique cDNAs, of which 77% were previously annotated. Of the 94 cDNAs encoding proteins of unknown function, 24% either had no match in databases or contained a secretory signal that could not be predicted from electronic data.     

Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis

Numerous studies have shown the presence of DNA strand breaks in human ejaculated spermatozoa. The nature of this nuclear anomaly and its relationship to patient etiology is however poorly understood. The aim of this study was to investigate the relationship between nuclear DNA damage, assessed using the TUNEL assay and a number of key apoptotic markers, including Fas, Bcl-x, and p53, in ejaculated human spermatozoa from men with normal and abnormal semen parameters. We also determined the nature of the DNA damage by examining the percentage of ejaculated spermatozoa exhibiting DNA damage using the comet assay and by challenging sperm chromatin to attack by micrococcal nuclease S7 and DNase I. We show that TUNEL positivity and apoptotic markers do not always exist in unison; however, semen samples that had a low sperm concentration and poor morphology were more likely to show high levels of TUNEL positivity and Fas and p53 expression. In addition, the DNA damage in ejaculated human sperm is represented by both single- and double-stranded DNA breaks, and access to the DNA is restricted by the compacted nature of ejaculated spermatozoa. This DNA protection is poorer in men with abnormal semen parameters. We propose that the presence of DNA damage is not directly linked to an apoptotic process occurring in spermatozoa and arises due to problems in the nuclear remodeling process. Subsequently, the presence of apoptotic proteins in ejaculated spermatozoa may be linked to defects in cytoplasmic remodeling during the later stages of spermatogenesis.     

Progesterone, but not progesterone-independent activation of progestin receptors by a mating stimulus, rapidly decreases progestin receptor immunoreactivity in female rat brain

Recent studies suggest that progestin receptors may be activated in vivo by neurotransmitters in the absence of ligand. More specifically, vaginal-cervical stimulation (VCS) can influence sexual behavior by activating progestin receptors in the absence of progesterone. Another way to test if progestin receptors are influenced by particular stimuli is to examine progestin receptor immunostaining. We report that progestin receptor immunoreactivity is decreased in the forebrain of estradiol-primed ovariectomized (OVX) rats within 1 h after a subcutaneous injection of progesterone, a time by which rapid down-regulation of progestin receptors does not seem to have occurred. In estradiol-primed OVX rats, VCS also decreased progestin receptor immunoreactivity within 1 h in the medial preoptic area, but not in any other area examined. To determine if the decrease in immunoreactivity by VCS was due to adrenal secretions or by ligand-independent activation of progestin receptors, we repeated the experiment in estradiol-primed OVX/adrenalectomized rats. Prior removal of the adrenal glands blocked the rapid decrease in progestin receptor immunoreactivity, even though data from other experiments suggest that progestin receptors are activated by VCS at this time. These studies suggest the possibility that progestin receptors may be affected differentially by progesterone-dependent or by progesterone-independent pathways. This raises the possibility that activation of progestin receptors by these two distinct pathways may lead to different neuronal consequences.     

B30.2-like domain proteins: update and new insights into a rapidly expanding family of proteins

The B30.2 domain is a conserved region of around 170 amino acids associated with several different protein domains, including the immunoglobulin folds of butyrophilin and the RING finger domain of ret finger protein. We recently reported several novel members of this family as well as previously undescribed protein families possessing the B30.2 domain. Many proteins have subsequently been found to possess this domain, including pyrin/marenostrin and the midline 1 (MID1) protein. Mutations in the B30.2 domain of pyrin/marenostrin are implicated in familial Mediterranean fever, and partial loss of the B30.2 domain of MID1 is responsible for Opitz G/BBB syndrome, characterized by developmental midline defects. In this study, we scrutinized the available sequence data bases for the identification of novel B30.2 domain proteins using highly sensitive database-searching tools. In addition, we discuss the chromosomal localization of genes in the B30.2 family, since the encoded proteins are likely to be involved in other forms of periodic fever, autoimmune, and genetic diseases.      

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

Placentation in dolphins from the Amazon River Basin: the Boto, Inia geoffrensis, and the Tucuxi, Sotalia fluviatilis

A recent reassessment of the phylogenetic affinities of cetaceans makes it timely to compare their placentation with that of the artiodactyls. We studied the placentae of two sympatric species of dolphin from the Amazon River Basin, representing two distinct families. The umbilical cord branched to supply a bilobed allantoic sac. Small blood vessels and smooth muscle bundles were found within the stroma of the cord. Foci of squamous metaplasia occurred in the allanto-amnion and allantochorion. The interhemal membrane of the placenta was of the epitheliochorial type. Two different types of trophoblastic epithelium were seen. Most was of the simple columnar type and indented by fetal capillaries. However, there were also areolar regions with tall columnar trophoblast and these were more sparsely supplied with capillaries. The endometrium was well vascularised and richly supplied with actively secreting glands. These findings are consistent with the current view that Cetacea are nested within Artiodactyla as sister group to the hippopotamids.     

Recessive Osteogenesis Imperfecta Caused by Missense Mutations in SPARC

Secreted protein, acidic, cysteine-rich (SPARC) is a glycoprotein that binds to collagen type I and other proteins in the extracellular matrix. Using whole-exome sequencing to identify the molecular defect in two unrelated girls with severe bone fragility and a clinical diagnosis of osteogenesis imperfecta type IV, we identified two homozygous variants in SPARC (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"NM_003118.3","term_id":"365777426","term_text":"NM_003118.3"}}NM_003118.3; c.497G>A [p.Arg166His] in individual 1; c.787G>A [p.Glu263Lys] in individual 2). Published modeling and site-directed mutagenesis studies had previously shown that the residues substituted by these mutations form an intramolecular salt bridge in SPARC and are essential for the binding of SPARC to collagen type I. The amount of SPARC secreted by skin fibroblasts was reduced in individual 1 but appeared normal in individual 2. The migration of collagen type I alpha chains produced by these fibroblasts was mildly delayed on SDS-PAGE gel, suggesting some overmodification of collagen during triple helical formation. Pulse-chase experiments showed that collagen type I secretion was mildly delayed in skin fibroblasts from both individuals. Analysis of an iliac bone sample from individual 2 showed that trabecular bone was hypermineralized on the material level. In conclusion, these observations show that homozygous mutations in SPARC can give rise to severe bone fragility in humans.     

Coxiella burnetii: turning hostility into a home

Coxiella burnetii, the causative agent of the human disease Q fever, is a unique intracellular bacterial pathogen. Coxiella replicates to high numbers within a pathogen‐derived lysosome‐like vacuole, thriving within a low pH, highly proteolytic and oxidative environment. In 2009, researchers developed means to axenically culture Coxiella paving the way for the development of tools to genetically manipulate the organism. These advances have revolutionized our capacity to examine the pathogenesis of Coxiella. In recent years, targeted and random mutant strains have been used to demonstrate that the Dot/Icm type IV secretion system is essential for intracellular replication of Coxiella. Current research is focused towards understanding the unique cohort of over 130 effector proteins that are translocated into the host cell. Mutagenesis screens have been employed to identify effectors that play important roles for the biogenesis of the Coxiella‐containing vacuole and intracellular replication of Coxiella. A surprisingly high number of effector mutants demonstrate significant intracellular growth defects, and future studies on the molecular function of these effectors will provide great insight into the pathogenesis of Coxiella. Already, this expanse of new data implicates many eukaryotic processes that are targeted by the arsenal of Coxiella effectors including autophagy, apoptosis and vesicular trafficking.     

Zinc induces disorder-to-order transitions in free and membrane-associated Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2: a solution CD and solid-state ATR-FTIR study

Dehydrins are intrinsically unstructured proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. Although their role is not completely understood, it has been suggested that they stabilize proteins and membrane structures during environmental stress and also sequester metals such as zinc. Here, we investigate two dehydrins (denoted as TsDHN-1 and TsDHN-2) from Thellungiella salsuginea. This plant is a crucifer that thrives in the Canadian sub-Arctic (Yukon Territory) where it grows on saline-rich soils and experiences periods of both extreme cold and drought. We show using circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopy that ordered secondary structure is induced and stabilized in these proteins, both in free and vesicle-bound form, by association with zinc. In membrane-associated form, both proteins have an increased proportion of β-strand conformation induced by the cation, in addition to the amphipathic α-helices formed by their constituent K-segments. These results support the hypothesis that dehydrins stabilize plant plasma and organellar membranes in conditions of stress, and further that zinc may be an important co-factor in stabilization. Whereas dehydrins in the cytosol of a plant cell undergoing dehydration or temperature stress form bulk hydrogels and remain primarily disordered, dehydrins with specific membrane- or protein-associations will have induced ordered secondary structures.     

Topochemical distribution of lignin and hydroxycinnamic acids in sugar-cane cell walls and its correlation with the enzymatic hydrolysis of polysaccharides

Background  

Lignin and hemicelluloses are the major components limiting enzyme infiltration into cell walls. Determination of the topochemical distribution of lignin and aromatics in sugar cane might provide important data on the recalcitrance of specific cells. We used cellular ultraviolet (UV) microspectrophotometry (UMSP) to topochemically detect lignin and hydroxycinnamic acids in individual fiber, vessel and parenchyma cell walls of untreated and chlorite-treated sugar cane. Internodes, presenting typical vascular bundles and sucrose-storing parenchyma cells, were divided into rind and pith fractions.