Location-dependent effects of inhibition on local spiking in pyramidal neuron dendrites

Cortical computations are critically dependent on interactions between pyramidal neurons (PNs) and a menagerie of inhibitory interneuron types. A key feature distinguishing interneuron types is the spatial distribution of their synaptic contacts onto PNs, but the location-dependent effects of inhibition are mostly unknown, especially under conditions involving active dendritic responses. We studied the effect of somatic vs. dendritic inhibition on local spike generation in basal dendrites of layer 5 PNs both in neocortical slices and in simple and detailed compartmental models, with equivalent results: somatic inhibition divisively suppressed the amplitude of dendritic spikes recorded at the soma while minimally affecting dendritic spike thresholds. In contrast, distal dendritic inhibition raised dendritic spike thresholds while minimally affecting their amplitudes. On-the-path dendritic inhibition modulated both the gain and threshold of dendritic spikes depending on its distance from the spike initiation zone. Our findings suggest that cortical circuits could assign different mixtures of gain vs. threshold inhibition to different neural pathways, and thus tailor their local computations, by managing their relative activation of soma- vs. dendrite-targeting interneurons.     

Systematic Identification of Rhythmic Genes Reveals camk1gb as a New Element in the Circadian Clockwork

A wide variety of biochemical, physiological, and molecular processes are known to have daily rhythms driven by an endogenous circadian clock. While extensive research has greatly improved our understanding of the molecular mechanisms that constitute the circadian clock, the links between this clock and dependent processes have remained elusive. To address this gap in our knowledge, we have used RNA sequencing (RNA–seq) and DNA microarrays to systematically identify clock-controlled genes in the zebrafish pineal gland. In addition to a comprehensive view of the expression pattern of known clock components within this master clock tissue, this approach has revealed novel potential elements of the circadian timing system. We have implicated one rhythmically expressed gene, camk1gb, in connecting the clock with downstream physiology of the pineal gland. Remarkably, knockdown of camk1gb disrupts locomotor activity in the whole larva, even though it is predominantly expressed within the pineal gland. Therefore, it appears that camk1gb plays a role in linking the pineal master clock with the periphery.     

H5-type influenza virus hemagglutinin is functionally recognized by the natural killer-activating receptor NKp44

Antiviral immune defenses involve natural killer (NK) cells. We previously showed that the NK-activating receptor NKp44 is involved in the functional recognition of H1-type influenza virus strains by NK cells. In the present study, we investigated the interaction of NKp44 and the hemagglutinin of a primary influenza virus H5N1 isolate. Here we show that recombinant NKp44 recognizes H5-expressing cells and specifically interacts with soluble H5 hemagglutinin. H5-pseudotyped lentiviral particles bind to NK cells expressing NKp44. Following interaction with target cells expressing H5, pseudotyped lentiviral particles, or membrane-associated H5, NK cells show NKp44-mediated induced activity. These findings indicate that NKp44-H5 interactions induce functional NK activation.     

Cells on the run: shear-regulated integrin activation in leukocyte rolling and arrest on endothelial cells

The arrest of rolling leukocytes on various target vascular beds is mediated by specialized leukocyte integrins and their endothelial immunoglobulin superfamily (IgSF) ligands. These integrins are kept in largely inactive states and undergo in situ activation upon leukocyte-endothelial contact by both biochemical and mechanical signals from flow-derived shear forces. In vivo and in vitro studies suggest that leukocyte integrin activation involves conformational alterations through inside-out signaling followed by ligand-induced rearrangements accelerated by external forces. This activation process takes place within fractions of seconds by in situ signals transduced to the rolling leukocyte as it encounters specialized endothelial-displayed chemoattractants, collectively termed arrest chemokines. In neutrophils, selectin rolling engagements trigger intermediate affinity integrins to support reversible adhesions before chemokine-triggered arrest. Different leukocyte subsets appear to use different modalities of integrin activation during rolling and arrest at distinct endothelial sites.     

Reciprocal changes in calcification of the gastrolith and cuticle during the molt cycle of the red claw crayfish Cherax quadricarinatus

Mobilization of calcium during the molt cycle from the cuticle to transient calcium deposits is widely spread in crustaceans. The dynamics of calcium transport to transient calcium deposits called gastroliths and to the cuticle over the course of the molt cycle were studied in the crayfish Cherax quadricarinatus. In this species, calcium was deposited in the gastroliths during premolt and transported back to the cuticle during postmolt, shown by digital X-ray radiograph analysis. The predominant mineral in the crayfish is amorphous calcium carbonate embedded in an organic matrix composed mainly of chitin. Scanning electron micrographs of the cuticle during premolt showed that the endocuticle and parts of the exocuticle were the source of most of the labile calcium, while the epicuticle did not undergo degradation and remained mineralized throughout the molt cycle. The gastroliths are made of concentric layers of amorphous calcium carbonate intercalated between chitinous lamella. Measurements of pH and calcium levels during gastrolith deposition showed that calcium concentrations in the gastroliths, stomach, and muscle were about the same (10 to 11 mmol l(-1)). On the other hand, pH varied greatly, from 8.7+/-0.15 in the gastrolith cavity through 7.6+/-0.2 in muscle to 6.9+/-0.5 in the stomach.     

Characterization of the Novel Fusobacterium nucleatum Plasmid pKH9 and Evidence of an Addiction System

Fusobacterium nucleatum is an important oral anaerobic pathogen involved in periodontal and systemic infections. Studies of the molecular mechanisms involved in fusobacterial virulence and adhesion have been limited by lack of systems for efficient genetic manipulation. Plasmids were isolated from eight strains of F. nucleatum. The smallest plasmid, pKH9 (4,975 bp), was characterized and used to create new vectors for fusobacterial genetic manipulation. DNA sequence analysis of pKH9 revealed an open reading frame (ORF) encoding a putative autonomous rolling circle replication protein (Rep), an ORF predicted to encode a protein homologous to members of the FtsK/SpoIIIE cell division-DNA segregation protein family, and an operon encoding a putative toxin-antitoxin plasmid addiction system (txf-axf). Deletion analysis localized the pKH9 replication region in a 0.96-kbp fragment. The pKH9 rep gene is not present in this fragment, suggesting that pKH9 can replicate in fusobacteria independently of the Rep protein. A pKH9-based, compact Escherichia coli-F. nucleatum shuttle plasmid was constructed and found to be compatible with a previously described pFN1-based fusobacterial shuttle plasmid. Deletion of the pKH9 putative addiction system (txf-axf) reduced plasmid stability in fusobacteria, indicating its addiction properties and suggesting it to be the first plasmid addiction system described for fusobacteria. pKH9, its genetic elements, and its shuttle plasmid derivatives can serve as useful tools for investigating fusobacterial properties important in biofilm ecology and pathogenesis.     

Human quiescin-sulfhydryl oxidase, QSOX1: probing internal redox steps by mutagenesis

The flavoprotein quiescin-sulfhydryl oxidase (QSOX) rapidly inserts disulfide bonds into unfolded, reduced proteins with the concomitant reduction of oxygen to hydrogen peroxide. This study reports the first heterologous expression and enzymological characterization of a human QSOX1 isoform. Like QSOX isolated from avian egg white, recombinant HsQSOX1 is highly active toward reduced ribonuclease A (RNase) and dithiothreitol but shows a >100-fold lower k cat/ K m for reduced glutathione. Previous studies on avian QSOX led to a model in which reducing equivalents were proposed to relay through the enzyme from the first thioredoxin domain (C70-C73) to a distal disulfide (C509-C512), then across the dimer interface to the FAD-proximal disulfide (C449-C452), and finally to the FAD. The present work shows that, unlike the native avian enzyme, HsQSOX1 is monomeric. The recombinant expression system enabled construction of the first cysteine mutants for mechanistic dissection of this enzyme family. Activity assays with mutant HsQSOX1 indicated that the conserved distal C509-C512 disulfide is dispensable for the oxidation of reduced RNase or dithiothreitol. The four other cysteine residues chosen for mutagenesis, C70, C73, C449, and C452, are all crucial for efficient oxidation of reduced RNase. C452, of the proximal disulfide, is shown to be the charge-transfer donor to the flavin ring of QSOX, and its partner, C449, is expected to be the interchange thiol, forming a mixed disulfide with C70 in the thioredoxin domain. These data demonstrate that all the internal redox steps occur within the same polypeptide chain of mammalian QSOX and commence with a direct interaction between the reduced thioredoxin domain and the proximal disulfide of the Erv/ALR domain.     

Esterase from the oil-degrading Acinetobacter lwoffii RAG-1: Sequence analysis and over-expression in Escherichia coli

Abstract The antigenic properties of the surface layer (S-layer) proteins of various Campylobacter rectus strains including 24 clinical isolates and the type strain ATCC 33238 were examined. S-layer proteins were extracted from whole cells by acid treatment according to the method of McCoy et al. (Infect. Immun. 11, 517–525, 1975). The acid extracts from 23 of the isolates and ATCC 33238 contained two major proteins with molecular masses of 130 kDa and 150 kDa, both of which were identified as subunits of the S-layer after comparison with the protein profiles of acid-treated (S-layer-deficient) cells. An S-layer protein from one isolate (CI-808) demonstrated a different molecular mass (160 kDa). Both the 150-kDa proteins of ATCC 33238 and isolate CI-306 and the 160-kDa protein of CI-808 were purified by ion-exchange chromatography in the presence of urea. In Ouchterlony immunodiffusion experiments with these purified proteins and rabbit antiserum raised to each purified protein, both common and strain-specific antigenic determinants were identified in the C. rectus S-layer proteins.