Neuregulin 3 promotes excitatory synapse formation on hippocampal interneurons

Hippocampal GABAergic interneurons are crucial for cortical network function and have been implicated in psychiatric disorders. We show here that Neuregulin 3 (Nrg3), a relatively little investigated low‐affinity ligand, is a functionally dominant interaction partner of ErbB4 in parvalbumin‐positive (PV) interneurons. Nrg3 and ErbB4 are located pre‐ and postsynaptically, respectively, in excitatory synapses on PV interneurons in vivo. Additionally, we show that ablation of Nrg3 results in a similar phenotype as the one described for ErbB4 ablation, including reduced excitatory synapse numbers on PV interneurons, altered short‐term plasticity, and disinhibition of the hippocampal network. In culture, presynaptic Nrg3 increases excitatory synapse numbers on ErbB4⁺ interneurons and affects short‐term plasticity. Nrg3 mutant neurons are poor donors of presynaptic terminals in the presence of competing neurons that produce recombinant Nrg3, and this bias requires postsynaptic ErbB4 but not ErbB4 kinase activity. Furthermore, when presented by non‐neuronal cells, Nrg3 induces postsynaptic membrane specialization. Our data indicate that Nrg3 provides adhesive cues that facilitate excitatory neurons to synapse onto ErbB4⁺ interneurons.     

Interspecies recombination between the penA genes of Neisseria meningitidis and commensal Neisseria species during the emergence of penicillin resistance in N. meningitidis: natural events and laboratory simulation.

The penicillin-binding protein 2 genes (penA) of penicillin-resistant Neisseria meningitidis have a mosaic structure that has arisen by the introduction of regions from the penA genes of Neisseria flavescens or Neisseria cinerea. Chromosomal DNA from both N. cinerea and N. flavescens could transform a penicillin-susceptible isolate of N. meningitidis to increased resistance to penicillin. With N. flavescens DNA, transformation to resistance was accompanied by the introduction of the N. flavescens penA gene, providing a laboratory demonstration of the interspecies recombinational events that we believe underlie the development of penicillin resistance in many meningococci in nature. Surprisingly, with N. cinerea DNA, the penicillin-resistant transformants did not obtain the N. cinerea penA gene. However, the region of the penA gene derived from N. cinerea in N. meningitidis K196 contained an extra codon (Asp-345A) which was not found in any of the four N. cinerea isolates that we examined and which is known to result in a decrease in the affinity of PBP 2 in gonococci.     

Cell Cl and transepithelial Na transport in toad urinary bladder

Relationships between short-circuit current (I _sc), cell Cl and the mechanism(s) of Cl accumulation in toad bladder epithelial cells were investigated. In serosal Cl-free gluconate Ringer, 80% of the cell Cl (measured by x-ray microanalysis) was lost over 30–60 min with an associated decrease in cell water content. Concomitantly, I _sc fell to 20% of its initial value within 10 min but then recovered to 45% of its initial value despite continued Cl loss. With the reintroduction of Cl, cell Cl and I _sc both recovered within 10 min. Serosal SITS (4 acetamido-4-isothiocyano-stilbene-2,2-disulfonate; 0.5 mm) plus bumetanide (0.1 mm), did not prevent the fall in I _sc or the loss of cell Cl in gluconate medium, although they did inhibit subsequent recovery of I _sc in this medium. They also prevented the recovery of I _sc in Cl medium but not the reaccumulation of Cl by the cells. Although SITS and bumetanide did not prevent the loss or recovery of Cl, they modified the pattern of the ion changes. In their absence, changes in cellular Cl were twice that of the changes in measured cellular cations implicating basolateral Cl/HCO₃ exchange in Cl movement. With SITS plus bumetanide present, changes of similar magnitude in Cl were associated with equivalent changes in cation, consistent with the inhibition of Cl/ HCO₃ exchange.This work was supported by a grant from the Medical Research Council of New Zealand. Purchase of equipment was made possible through grants from the Medical Research Council of New Zealand, the Medical Distribution Committee of the Lottery Board, the University Grants Committee, the Telford Trust, the New Zealand Neurological Foundation and the National Heart Foundation. The expert technical assistance of S. Zellhuber-McMillan is gratefully acknowledged.     

The effect of in vivo heat treatments on blowfly flight muscle mitochondrial function: Effects on partial reactions of the respiratory chain

1. 1. The development of thermotolerance has been shown to protect blowfly flight muscle mitochondrial function from damage resulting from an LD₅₀ in vivo heat dose.

2. 2. The principal sites of the damage have been studied using specific inhibitors of the respiratory chain, rotenone and antimycin A, together with substrates that stimulate respiration through the different complexes.

3. 3. Complex I was identified as the primary site for heat damage. State III respiration was inhibited following the LD₅₀ in vivo heat dose, and uncoupling with FCCP did not restore respiration to control levels, indicating that the respiratory enzymes were inactivated. The development of thermotolerance protected this site from heat damage.

4. 4. In contrast, G3-P stimulated respiration was the same in control, LD₅₀ in vivo treated controls and LD₅₀, in vivo treated thermotolerant mitochondria, and significantly higher than state III respiration of LD₅₀ in vivo treated controls. This suggested that respiration through G3-P dehydrogenase, Co enzyme Q and Complex III is not damaged. However, as G3-P stimulated respiration of coupled mitochondria from LD₅₀ in-vivo treated flies was markedly reduced (El-Wadawi and Bowler, 1995. J. exp. Biol. 198: 2413–2421), phosphorylation at complex III may be inhibited also.

5. 5. Ferrocyanide stimulated respiration through cytochrome c-Complex IV was also inhibited in LD₅₀ in vivo treated flies, as compared with unheated control mitochondria. However, thermotolerance protected this site also from heat damage.

     

Pen and Pal Are Nucleotide-Sugar Dehydratases That Convert UDP-GlcNAc to UDP-6-Deoxy-d-GlcNAc-5,6-ene and Then to UDP-4-Keto-6-deoxy-l-AltNAc for CMP-Pseudaminic Acid Synthesis in Bacillus thuringiensis

CMP-pseudaminic acid is a precursor required for the O-glycosylation of flagellin in some pathogenic Gram-negative bacteria, a process known to be critical in bacterial motility and infection. However, little is known about flagellin glycosylation in Gram-positive bacteria. Here, we identified and functionally characterized an operon, named Bti_pse, in Bacillus thuringiensis israelensis ATCC 35646, which encodes seven different enzymes that together convert UDP-GlcNAc to CMP-pseudaminic acid. In contrast, Gram-negative bacteria complete this reaction with six enzymes. The first enzyme, which we named Pen, converts UDP-d-GlcNAc to an uncommon UDP-sugar, UDP-6-deoxy-d-GlcNAc-5,6-ene. Pen contains strongly bound NADP⁺ and has distinct UDP-GlcNAc 4-oxidase, 5,6-dehydratase, and 4-reductase activities. The second enzyme, which we named Pal, converts UDP-6-deoxy-d-GlcNAc-5,6-ene to UDP-4-keto-6-deoxy-l-AltNAc. Pal is NAD⁺-dependent and has distinct UDP-6-deoxy-d-GlcNAc-5,6-ene 4-oxidase, 5,6-reductase, and 5-epimerase activities. We also show here using NMR spectroscopy and mass spectrometry that in B. thuringiensis, the enzymatic product of Pen and Pal, UDP-4-keto-6-deoxy-l-AltNAc, is converted to CMP-pseudaminic acid by the sequential activities of a C4″-transaminase (Pam), a 4-N-acetyltransferase (Pdi), a UDP-hydrolase (Phy), an enzyme (Ppa) that adds phosphoenolpyruvate to form pseudaminic acid, and finally a cytidylyltransferase that condenses CTP to generate CMP-pseudaminic acid. Knowledge of the distinct dehydratase-like enzymes Pen and Pal and their role in CMP-pseudaminic acid biosynthesis in Gram-positive bacteria provides a foundation to investigate the role of pseudaminic acid and flagellin glycosylation in Bacillus and their involvement in bacterial motility and pathogenicity.     

Sequence composition effects on denatured state loop formation in iso-1-cytochrome c variants: polyalanine versus polyglycine inserts

Protein folding is dependent on the formation and persistence of simple loops during the earliest events of the folding process. Ease of loop formation and persistence is believed to be dependent on the steric properties of the residues involved in loop formation. We have investigated this conformational factor in the denatured state of iso-1-cytchrome c using a five alanine insert in front of a unique histidine in the N-terminal region of the protein. The alanine residues have then been progressively substituted with sterically less-constrained glycine residues. Guanidine-HCl unfolding shows that all variants have a free energy of unfolding of approximately 2 kcal/mol. The low stability of these variants is well accounted for by stabilization of the denatured state by histidine-heme loop formation. The stability of the 22 residue histidine-heme loop has been measured in 3 M guanidine hydrochloride for all variants. Surprisingly, relative to alanine, glycine has only a very modest effect on equilibrium loop stability. Thus, the greater flexibility that glycine confers on the main-chain provides no advantage in terms of the persistence of simple loops early in folding. The underlying basis for the similar behavior of loops with polyalanine versus polyglycine inserts is discussed in terms of the current knowledge of the structure and loop formation kinetics of glycine versus alanine-rich peptides.     

Adult Non-Cystic Fibrosis Bronchiectasis Is Characterised by Airway Luminal Th17 Pathway Activation

Background

Non-cystic fibrosis (CF) bronchiectasis is characterised by chronic airway infection and neutrophilic inflammation, which we hypothesised would be associated with Th17 pathway activation.

Methods

Th17 pathway cytokines were quantified in bronchoalveolar lavage fluid (BALF), and gene expression of IL-17A, IL-1β, IL-8 and IL-23 determined from endobronchial biopsies (EBx) in 41 stable bronchiectasis subjects and 20 healthy controls. Relationships between IL-17A levels and infection status, important clinical measures and subsequent Pseudomonas aeruginosa infection were determined.

Results

BALF levels of all Th17 cytokines (median (IQR) pg/mL) were significantly higher in bronchiectasis than control subjects, including IL-17A (1.73 (1.19, 3.23) vs. 0.27 (0.24, 0.35), 95% CI 1.05 to 2.21, p<0.0001) and IL-23 (9.48 (4.79, 15.75) vs. 0.70 (0.43, 1.79), 95% CI 4.68 to 11.21, p<0.0001). However, BALF IL-17A levels were not associated with clinical measures or airway microbiology, nor predictive of subsequent P. aeruginosa infection. Furthermore, gene expression of IL-17A in bronchiectasis EBx did not differ from control. In contrast, gene expression (relative to medians of controls) in bronchiectasis EBx was significantly higher than control for IL1β (4.12 (1.24, 8.05) vs 1 (0.13, 2.95), 95% CI 0.05 to 4.07, p = 0.04) and IL-8 (3.75 (1.64, 11.27) vs 1 (0.54, 3.89), 95% CI 0.32 to 4.87, p = 0.02) and BALF IL-8 and IL-1α levels showed significant relationships with clinical measures and airway microbiology. P. aeruginosa infection was associated with increased levels of IL-8 while Haemophilus influenzae was associated with increased IL-1α.

Conclusions and Clinical Relevance

Established adult non-CF bronchiectasis is characterised by luminal Th17 pathway activation, however this pathway may be relatively less important than activation of non-antigen-specific innate neutrophilic immunity.     

A study of reproductive females of the freshwater crayfish Austropotamobius pallipes

Details of the female annual reproductive cycle in the population from Northumberland are given. Mating occurs in October and is followed by egg laying in early November. Eggs are carried by females until the following July–August when hatching occurs. The smallest berried female caught was 25 mm carapace length (C.L.) and this was chosen as the minimum size for adult females. Only 40–50% of adult females, hand-caught, were berried in the Spring following egg laying; but this percentage was low largely because few females in the size classes below 28 mm C.L. were berried. The reasons for adult females being non-reproductive in a year are discussed.     

Modulation of platinum antitumor drug binding to DNA by linked and free intercalators

We report the DNA binding site preferences of the novel molecule AO-Pt, in which the anticancer drug dichloro(ethylenediamine)platinum(II) is linked by a hexamethylene chain to acridine orange. The sequence specificity of platinum binding was mapped by exonuclease III digestion of 165 and 335 base pair restriction fragments from pBR322 DNA. Parallel studies were carried out with the unmodified anticancer drugs cis-diamminedichloroplatinum(II) (cis-DDP) and dichloro(ethylenediamine)platinum(II), [Pt(en)Cl2]. Oligo(dG) sequences are the most prevalent binding sites for AO-Pt, with secondary binding occurring mainly at d(AG) sites. cis-DDP and [Pt(en)Cl2] bind less readily to the secondary sequences, with cis-DDP showing greater binding site selectivity than [Pt(en)Cl2]. The DNA intercalator ethidium bromide promotes binding of [Pt(en)Cl2] and cis-DDP to many sites containing d(CGG) and, to a lesser extent, d(AG) sequences. AO-Pt exhibits enhanced binding to these sequences without the need for an external intercalator. Unlinked acridine orange, however, does not promote binding of [Pt(en)Cl2] and cis-DDP to d(CGG) and d(AG) sequences. These results are discussed in terms of the sequence preferences, stereochemistry, and relative residence times of the intercalators at their DNA binding sites. By modulating local structure in a sequence-dependent manner, both linked and, in the case of ethidium, free intercalators can influence the regioselectivity of covalent modification of DNA by platinum antitumor drugs.     

Rat brain (Na+-K+)ATPase: modulation of its ouabain-sensitive K+-PNPPase activity by thimerosal

1. The (Na+ + K+) ATPase activity of a rat brain synaptic membrane preparation was inhibited by 10(-5) M thimerosal. 2. The ouabain inhibitable K+-PNPPase activity of thimerosal treated membranes was compared with that of untreated membranes with respect to sensitivity to temperature, ouabain, K+ and ATP. 3. All those kinetic characteristics were substantially altered by treatment with thimerosal.