Simultaneous knockout of Slo3 and CatSper1 abolishes all alkalization- and voltage-activated current in mouse spermatozoa

During passage through the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that renders sperm competent to produce fertilization. Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg. In mouse sperm, the development of hyperactivated motility is dependent on cytosolic alkalization that then results in an increase in cytosolic Ca²⁺. The elevation of Ca²⁺ is thought to be primarily driven by the concerted interplay of two alkalization-activated currents, a K⁺ current (KSPER) composed of pore-forming subunits encoded by the Kcnu1 gene (also termed Slo3) and a Ca²⁺ current arising from a family of CATSPER subunits. After deletion of any of four CATSPER subunit genes (CATSPER1–4), the major remaining current in mouse sperm is alkalization-activated KSPER current. After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K⁺ current hypothesized to reflect monovalent flux through CATSPER. Here, we address two questions. First, does the residual outward K⁺ current present in the Slo3 ^−/− sperm arise from CATSPER? Second, can any additional membrane K⁺ currents be detected in mouse sperm by patch-clamp methods other than CATSPER and KSPER? Here, using mice bred to lack both SLO3 and CATSPER1 subunits, we show conclusively that the voltage-activated outward current present in Slo3 ^−/− sperm is abolished when CATSPER is also deleted. Any leak currents that may play a role in setting the resting membrane potential in noncapacitated sperm are likely smaller than the pipette leak current and thus cannot be resolved within the limitation of the patch-clamp technique. Together, KSPER and CATSPER appear to be the sole ion channels present in mouse sperm that regulate membrane potential and Ca²⁺ influx in response to alkalization.     

Extracellular Signal-regulated Kinase 5 (ERK5) Mediates Prolactin-stimulated Adult Neurogenesis in the Subventricular Zone and Olfactory Bulb

Prolactin-stimulated adult neurogenesis in the subventricular zone (SVZ) and olfactory bulb (OB) mediates several reproductive behaviors including mating/pregnancy, dominant male pheromone preference in females, and paternal recognition of offspring. However, downstream signaling mechanisms underlying prolactin-induced adult neurogenesis are completely unknown. We report here for the first time that prolactin activates extracellular signal-regulated kinase 5 (ERK5), a MAP kinase that is specifically expressed in the neurogenic regions of the adult mouse brain. Knockdown of ERK5 by retroviral infection of shRNA attenuates prolactin-stimulated neurogenesis in SVZ-derived adult neural stem/progenitor cells (aNPCs). Inducible erk5 deletion in adult neural stem cells of transgenic mice inhibits neurogenesis in the SVZ and OB following prolactin infusion or mating/pregnancy. These results identify ERK5 as a novel and critical signaling mechanism underlying prolactin-induced adult neurogenesis.     

Altered Intersubunit Communication Is the Molecular Basis for Functional Defects of Pathogenic p97 Mutants

The human AAA ATPase p97 is a molecular chaperone essential in cellular proteostasis. Single amino acid substitutions in p97 have been linked to a clinical multiple-disorder condition known as inclusion body myopathy associated with Paget''s disease of the bone and frontotemporal dementia. How the mutations affect the molecular mechanism that governs the function of p97 remains unclear. Here, we show that within the hexameric ring of a mutant p97, D1 domains fail to regulate their respective nucleotide-binding states, as evidenced by the lower amount of prebound ADP, weaker ADP binding affinity, full occupancy of adenosine-5′-O-(3-thiotriphosphate) binding, and elevated overall ATPase activity, indicating a loss of communication among subunits. Defective communication between subunits is further illustrated by altered conformation in the side chain of residue Phe-360 that probes into the nucleotide-binding pocket from a neighboring subunit. Consequently, conformations of N domains in a hexameric ring of a mutant p97 become uncoordinated, thus impacting its ability to process substrate.     

Regulation of a Dynamic Interaction between Two Microtubule-binding Proteins,EB1 and TIP150, by the Mitotic p300/CBP-associated Factor (PCAF) Orchestrates Kinetochore Microtubule Plasticity and Chromosome Stability during Mitosis

The microtubule cytoskeleton network orchestrates cellular dynamics and chromosome stability in mitosis. Although tubulin acetylation is essential for cellular plasticity, it has remained elusive how kinetochore microtubule plus-end dynamics are regulated by p300/CBP-associated factor (PCAF) acetylation in mitosis. Here, we demonstrate that the plus-end tracking protein, TIP150, regulates dynamic kinetochore-microtubule attachments by promoting the stability of spindle microtubule plus-ends. Suppression of TIP150 by siRNA results in metaphase alignment delays and perturbations in chromosome biorientation. TIP150 is a tetramer that binds an end-binding protein (EB1) dimer through the C-terminal domains, and overexpression of the C-terminal TIP150 or disruption of the TIP150-EB1 interface by a membrane-permeable peptide perturbs chromosome segregation. Acetylation of EB1-PCAF regulates the TIP150 interaction, and persistent acetylation perturbs EB1-TIP150 interaction and accurate metaphase alignment, resulting in spindle checkpoint activation. Suppression of the mitotic checkpoint serine/threonine protein kinase, BubR1, overrides mitotic arrest induced by impaired EB1-TIP150 interaction, but cells exhibit whole chromosome aneuploidy. Thus, the results identify a mechanism by which the TIP150-EB1 interaction governs kinetochore microtubule plus-end plasticity and establish that the temporal control of the TIP150-EB1 interaction by PCAF acetylation ensures chromosome stability in mitosis.     

Pyrosequencing Analysis of the Salivary Microbiota of Healthy Chinese Children and Adults

Describing the biogeography of bacterial communities within the human body is critical for establishing healthy baselines from which to detect differences associated with diseases. Little is known, however, about the baseline of normal salivary microbiota from healthy Chinese children and adults. With parallel barcoded 454 pyrosequencing, the bacterial diversity and richness of saliva were thoroughly investigated from ten healthy Chinese children and adults. The overall taxonomic distribution of our metagenomic data demonstrated that the diversity of salivary microbiota from children was more complex than adults, while the composition and richness of salivary microbiota were similar in children and adults, especially for predominant bacteria. A large number of bacterial phylotypes were shared by healthy children and adults, indicating the existence of a core salivary microbiome. In children and adults, the vast majority of sequences in salivary microbiota belonged to Streptococcus, Prevotella, Neisseria, Haemophilus, Porphyromonas, Gemella, Rothia, Granulicatella, Fusobacterium, Actinomyces, Veillonella, and Aggregatibacter, which constituted the major components of normal salivary microbiota. With the exception of Actinomyces, the other seven non-predominant bacteria including Moraxella, Leptotrichia, Peptostreptococcus, Eubacterium, and members of Neisseriaceae, Flavobacteriaceae, and SR1 showed significant differences between children and adults (p?<?0.05). We first established the framework of normal salivary microbiota from healthy Chinese children and adults. Our data represent a critical step for determining the diversity of healthy microbiota in Chinese children and adults, and our data established a platform for additional large-scale studies focusing on the interactions between health and diseases in the future.     

Molecular mapping of leaf rust resistance gene LrNJ97 in Chinese wheat line Neijiang 977671

Neijiang 977671 and 19 near-isogenic lines with known leaf rust resistance genes were inoculated with 12 pathotypes of Puccinia triticina for postulation of leaf rust resistance genes effective at the seedling stage. The reaction pattern of Neijiang 977671 differed from those of the lines with known leaf rust resistance genes used in the test, indicating that Neijiang 977671 may carry a new leaf rust resistance gene(s). With the objective of identifying and mapping the new gene for resistance to leaf rust, F₁ and F₂ plants, and F_2:3 families, from Neijiang 977671 × Zhengzhou 5389 (susceptible) were inoculated with Chinese P. triticina pathotype FHNQ in the greenhouse. Results from the F₂ and F_2:3 populations indicated that a single dominant gene, temporarily designated LrNJ97, conferred resistance. In order to identify other possible genes in Neijiang 977671 other eight P. triticina pathotypes avirulent on Neijiang 977671 were used to inoculate 25 F_2:3 families. The results showed that at least three leaf rust resistance genes were deduced in Neijiang 977671. Bulked segregant analysis was performed on equal amounts of genomic DNA from 20 resistant and 20 susceptible F₂ plants. SSR markers polymorphic between the resistant and susceptible bulks were used to analyze the F_2:3 families. LrNJ97 was linked to five SSR loci on chromosome 2BL. The two closest flanking SSR loci were Xwmc317 and Xbarc159 at genetic distances of 4.2 and 2.2 cM, respectively. At present two designated genes (Lr50 and Lr58) are located on chromosome 2BL. In the seedling tests, the reaction pattern of LrNJ97 was different from that of Lr50. Lr50 and Lr58 were derived from T. armeniacum and Ae. triuncialis, respectively, whereas according to the pedigree of Neijiang 977671 LrNJ97 is from common wheat. Although seeds of lines with Lr58 were not available, it was concluded that LrNJ97 is likely to be a new leaf rust resistance gene.     

Tracing the Origin and Diversification of Dipodoidea (Order: Rodentia): Evidence from Fossil Record and Molecular Phylogeny

Dipodoidea are a diverse rodent group whose earliest known record is from the Middle Eocene. The evolution and diversification of this superfamily have been documented by fossils and comparative morphology, but have not yet been studied from the perspective of molecular phylogeny. This study is the first attempt to reconstruct an extensive phylogeny of Dipodidae and estimate divergence times based on a nuclear gene coding for interphotoreceptor retinoid-binding protein. We found that there is a wide measure of agreement with the fossil record. Each of the three ecological groups of the extant Dipodoidea (sicistines, zapodines, and jerboas) has its distinctive distribution; the distribution patterns have been shaped by the dispersal events. The key events of paleogeographic distribution coincided with major paleoenvironmental events in the Cenozoic. The first important diversification phase occurred during the period from the Oligocene to Early Miocene, when global climate underwent major changes beginning with the Eocene/Oligocene boundary. The second adaptive radiation occurred within jerboas and was associated with the expansion of open habitat starting with the late Middle Miocene. The diversification of jerboas can be correlated with habitat changes in response to global and regional climatic events.     

Comparison of the Genome Sequences of “Candidatus Portiera aleyrodidarum” Primary Endosymbionts of the Whitefly Bemisia tabaci B and Q Biotypes

“Candidatus Portiera aleyrodidarum” is the primary endosymbiont of whiteflies. We report two complete genome sequences of this bacterium from the worldwide invasive B and Q biotypes of the whitefly Bemisia tabaci. Differences in the two genome sequences may add insights into the complex differences in the biology of both biotypes.     

Molecular cloning and characterization of the mitogen-activated protein kinase kinase gene (MKK4) and its promoter sequence from oilseed rape (Brassica campestris L.)

Mitogen-activated protein kinase (MAPK) cascades are involved in various processes, including plant growth and development as well as biotic and abiotic stress responses. MAPK kinases (MKKs), which link MPKs and MAPKK kinases (MKKKs), are crucial in MAPK cascades because these kinases mediate various stress responses in plants. However, only few MKKs in Brassica campestris (rape) have been functionally characterized. In this study, a novel gene, MKK4 that belongs to a C MKK group, was isolated and characterized from rape. Bioinformatics analysis revealed that the length of cDNA was 1,317 bp with an open reading frame of 993 bp, which encodes a polypeptide containing 330 amino acids, including a putative signal peptide with 27 amino acid residues and a mature protein with 303 amino acids. The obtained MKK4 exhibited a predicted molecular mass of 36.5 kDa and an isoelectric point of 9.01. Quantitative real-time polymerase chain reaction analysis revealed that MKK4 expression could be induced by cold and salt. We also found that the MKK4 protein is localized in the nucleus. In addition, a 999 bp promoter fragment of MKK4 was cloned. Sequence analysis revealed that several putative regulatory elements were found in the MKK4 promoter. Transient expression assay showed that the MKK4 promoter fragments exhibited promoter activity and stimulated GFP expression. The effects of GFP gene expression at different temperatures and in different onion epidermis culture patterns were compared. Results showed that the MKK4 promoter could respond to low temperature and salt stress. These results suggested that MKK4 is possibly important for the regulation of cold- and salt-stress responses in plants.