Young dentate granule cells mediate pattern separation, whereas old granule cells facilitate pattern completion

Adult-born granule cells (GCs), a minor population of cells in the hippocampal dentate gyrus, are highly active during the first few weeks after functional integration into the neuronal network, distinguishing them from less active, older adult-born GCs and the major population of dentate GCs generated developmentally. To ascertain whether young and old GCs perform distinct memory functions, we created a transgenic mouse in which output of old GCs was specifically inhibited while leaving a substantial portion of young GCs intact. These mice exhibited enhanced or normal pattern separation between similar contexts, which was reduced following ablation of young GCs. Furthermore, these mutant mice exhibited deficits in rapid pattern completion. Therefore, pattern separation requires adult-born young GCs but not old GCs, and older GCs contribute to the rapid recall by pattern completion. Our data suggest that as adult-born GCs age, their function switches from pattern separation to rapid pattern completion.     

Mitogen-activated protein kinase phosphatase 2, MKP-2, regulates early inflammation in acute lung injury

Acute lung injury (ALI) is mediated by an early proinflammatory response resulting from either a direct or indirect insult to the lung mediating neutrophil infiltration and consequent disruption of the alveolar capillary membrane ultimately leading to refractory hypoxemia. The mitogen-activated protein kinase (MAPK) pathways are a key component of the molecular response activated by those insults triggering the proinflammatory response in ALI. The MAPK pathways are counterbalanced by a set of dual-specific phosphatases (DUSP) that deactivate the kinases by removing phosphate groups from tyrosine or threonine residues. We have previously shown that one DUSP, MKP-2, regulates the MAPK pathway in a model of sepsis-induced inflammation; however, the role of MKP-2 in modulating the inflammatory response in ALI has not been previously investigated. We utilized both MKP-2-null (MKP-2(-/-)) mice and MKP-2 knockdown in a murine macrophage cell line to elucidate the role of MKP-2 in regulating inflammation during ALI. Our data demonstrated attenuated proinflammatory cytokine production as well as decreased neutrophil infiltration in the lungs of MKP-2(-/-) mice following direct, intratracheal LPS. Importantly, when challenged with a viable pathogen, this decrease in neutrophil infiltration did not impact the ability of MKP-2(-/-) mice to clear either gram-positive or gram-negative bacteria. Furthermore, MKP-2 knockdown led to an attenuated proinflammatory response and was associated with an increase in phosphorylation of ERK and induction of a related DUSP, MKP-1. These data suggest that altering MKP-2 activity may have therapeutic potential to reduce lung inflammation in ALI without impacting pathogen clearance.     

ZyFISH: a simple, rapid and reliable zygosity assay for transgenic mice

Microinjection of DNA constructs into fertilized mouse oocytes typically results in random transgene integration at a single genomic locus. The resulting transgenic founders can be used to establish hemizygous transgenic mouse lines. However, practical and experimental reasons often require that such lines be bred to homozygosity. Transgene zygosity can be determined by progeny testing assays which are expensive and time-consuming, by quantitative Southern blotting which is labor-intensive, or by quantitative PCR (qPCR) which requires transgene-specific design. Here, we describe a zygosity assessment procedure based on fluorescent in situ hybridization (zyFISH). The zyFISH protocol entails the detection of transgenic loci by FISH and the concomitant assignment of homozygosity using a concise and unbiased scoring system. The method requires small volumes of blood, is scalable to at least 40 determinations per assay, and produces results entirely consistent with the progeny testing assay. This combination of reliability, simplicity and cost-effectiveness makes zyFISH a method of choice for transgenic mouse zygosity determinations.     

Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry

Capillary isoelectric focusing (CIEF) was used to profile the cellulase composition in complex fermentation samples of secreted proteins from Trichoderma reesei. The enzyme cellobiohydrolase I (CBH I, also referred to as Cel7A), a major component in these extracts, was purified from different strains and characterized using analytical methods such as CIEF, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD), and capillary liquid chromatography–electrospray mass spectrometry (cLC–ESMS). ESMS was also used to monitor the extent of glycosylation in CBH I isolated from T. reesei strain RUT-C30 and two derivative mutant strains. Selective identification of tryptic N-linked glycopeptides was achieved using LC–ESMS on a quadrupole/time-of-flight instrument with a mixed scan function. The suspected glycopeptides were further analyzed by on-line tandem mass spectrometry to determine the nature of N-linked glycans and their attachment sites. This strategy enabled the identification of a high mannose glycan attached to Asn270 (predominantly Man₈GlcNAc₂) and single GlcNAc occupancy at Asn45 and Asn384 with some site heterogeneity depending on strains and fermentation conditions. The linker region of CBH I was shown to be extensively glycosylated with di-, and tri-saccharides at Thr and Ser residues as indicated by MALDI-TOF and HPAEC–PAD experiments. Additional heterogeneity was noted in the CBH I linker peptide of RUT-C30 strain with the presence of a phosphorylated di-saccharide.     

Isolation of cadmium sensitive mutants in Chlamydomonas reinhardtii by transformation/insertional mutagenesis

Abstract An arg7, cw15, mt⁺ strain of Chlamydomonas reinhardtii (CC1618) was transformed with pARG7.8, a plasmid containing the wild-type ARG7 gene. Over 2300 arg⁺ transformants were selected on TAP media. Upon subsequent analysis on TAP plus cadmium plates, five of the transformants failed to grow at a level of 400 μM cadmium and were designated as cadmium sensitive (Cd^s) mutants. Hybridization data indicated that vector (pBR329) sequences were present in these five mutants, but not in the untransformed parental strain. Two of the mutants have been back crossed to an arg7, cw15, Cd⁺, mt⁻ strain (CC425) and found to have progeny which always cosegregate the arg⁺ and Cd^s phenotypesin these two mutants results from the insertion of the plasmid pARG7.8 into a gene involving cadmium detoxification, and it provides a method by which to clone the interrupted gene(s).     

Cryptic species and colonization processes in Ophryotrocha (Annelida,Dorvilleidae) inhabiting vertebrate remains in the shallow‐water Mediterranean

Over the past several years, there has been growing interest in how bones of decaying mammals are colonized in the marine seabed. One of the most common opportunistic taxa occurring worldwide on bones is dorvilleid polychaetes of the genus Ophryotrocha. In a recent study in the Mediterranean, Ophryotrocha puerilis and Ophryotrocha alborana were two of the most abundant species occurring in experimentally deployed bones. These species have direct development and this makes them a suitable model to study the mechanisms and processes allowing organisms lacking a dispersive larval phase to colonize new substrates. Here, we address the colonization processes at the molecular level for populations of O. puerilis and O. alborana on experimentally deployed mammal bones in the shallow‐water Mediterranean collected over a year at 3‐month intervals. High genetic distances between some of the O. puerilis organisms collected indicated the occurrence of at least two cryptic sibling species (O. puerilis ‘Shallow’ and O. puerilis ‘Deep’) apart from O. puerilis sensu stricto. This was corroborated with phylogenetic analyses using an alignment of three concatenated genes (COI, 16S, H3) and with species delimitation analyses using COI. The haplotype network inferred from COI sequences for O. puerilis ‘Shallow’ showed a few common haplotypes shared between the two trimesters analysed and several other less represented haplotypes only present in one trimester. Thus, colonization of these experimental bones may have been achieved by a few organisms that arrived to the bones and were able to reseed, and by several individuals arriving to the experimental bones and not persisting across time. In contrast, the haplotype network for O. alborana revealed that none of the haplotypes present in three different trimesters were shared, suggesting that the populations arriving at the bones during each trimester were totally replaced by new individuals during the subsequent trimesters. Our study suggests that different species of shallow‐water Ophryotrocha occurring in the Mediterranean may have different patterns of substrate colonization despite sharing similar life histories.     

Novel ways to prevent proteolysis — prophytepsin and proplasmepsin II

The recently determined crystal structures of two aspartic proteinase zymogens, prophytepsin from barley and proplasmepsin II from the malarial parasite Plasmodium falciparum, have provided new insights into zymogen inactivation. Prophytepsin shows a variation of the mechanism of inhibition used by the well-known gastric aspartic proteinase zymogens, whereas proplasmepsin II uses a completely new mode of inactivation.     

C-1027-induced alterations in Epstein-Barr viral DNA replication in latently infected cultured human Raji cells: relationship to DNA damage.

This study is the first detailing drug-induced changes in EBV DNA replication intermediates (RIs). Both EBV replication inhibition and damage induction were studied in latently infected human Raji cells treated with the enediyne DNA strand-scission agent C-1027. Analysis of RIs on two-dimensional agarose gels revealed a rapid loss in the EBV bubble arc. When elongation of nascent chains was blocked by aphidicolin, this loss was inhibited, suggesting that C-1027-induced disappearance of RIs was related to maturation of preformed replication molecules in the absence of initiation of new RIs. C-1027 damage to EBV DNA was limited at concentrations where loss of the bubble arc was nearly complete, and none was detected within the replicating origin (ori P)-containing fragment, indicating that replication inhibition occurred in trans. By contrast, the non-nuclear mitochondrial genome was insensitive to replication inhibition but highly sensitive to damage induced by C-1027. C-1027-induced trans inhibition of nuclear but not mitochondrial DNA replication is consistent with a cell cycle checkpoint response to a DNA-damaging agent. EBV replication and Raji cell growth were inhibited at equivalent C-1027 doses.