Availability of a diversely avid CD8+ T cell repertoire specific for the subdominant HLA-A2-restricted HIV-1 Gag p2419-27 epitope

HLA-A2-restricted CTL responses to immunodominant HIV-1 epitopes do not appear to be very effective in the control of viral replication in vivo. In this study, we studied human CD8+ T cell responses to the subdominant HLA-A2-restricted epitope TV9 (Gag p24(19-27), TLNAWVKVV) to explore the possibility of increasing its immune recognition. We confirmed in a cohort of 313 patients, infected by clade B or clade C viruses, that TV9 is rarely recognized. Of interest, the functional sensitivity of the TV9 response can be relatively high. The potential T cell repertoires for TV9 and the characteristics of constituent clonotypes were assessed by ex vivo priming of circulating CD8+ T cells from healthy seronegative donors. TV9-specific CTLs capable of suppressing viral replication in vitro were readily generated, suggesting that the cognate T cell repertoire is not limiting. However, these cultures contained multiple discrete populations with a range of binding avidities for the TV9 tetramer and correspondingly distinct functional dependencies on the CD8 coreceptor. The lack of dominant clonotypes was not affected by the stage of maturation of the priming dendritic cells. Cultures primed by dendritic cells transduced to present endogenous TV9 were also incapable of clonal maturation. Thus, a diffuse TCR repertoire appeared to be an intrinsic characteristic of TV9-specific responses. These data indicate that subdominance is not a function of poor immunogenicity, cognate TCR repertoire availability, or the potential avidity properties thereof, but rather suggest that useful responses to this epitope are suppressed by competing CD8+ T cell populations during HIV-1 infection.     

Soil organic carbon pools and productivity relationships for a 34 year old rice–wheat–jute agroecosystem under different fertilizer treatments

Soil organic carbon (SOC) pools are important in maintaining soil productivity and influencing the CO₂ loading into the atmosphere. An attempt is made here to investigate into the dynamics of pools of SOC viz., total organic carbon (C _tot), oxidisable organic carbon (C _oc) and its four different fractions such as very labile (C _{frac 1}), labile (C _{frac 2}), less labile (C _{frac 3}) and non-labile (C _{frac 4}), microbial biomass carbon (C _mic), mineralizable carbon (C _min), and particulate organic carbon (C _p) in relation to crop productivity using a 34 year old rice (Oryza sativa L)–wheat (Triticum aestivum L)–jute (Corchorus olitorius L) cropping system with different management strategies (no fertilization, only N, NP, NPK and NPK + FYM) in the hot humid, subtropics of India. A fallow treatment was also included to compare the impact of cultivation vis-à-vis no cultivation. Cultivation over the years caused a net decrease, while balanced fertilization with NPK maintained the SOC pools at par with the fallow. Only 22% of the C applied as FYM was stabilized into SOC, while the rest got lost. Of the analysed pools, C _{frac 1}, C _mic, C _p and C _min were influenced most by the treatments imposed. Most of the labile pools were significantly correlated with each other and with the yield and sustainable yield index (SYI) of the studied system. Of them, C _frac1, C _min, C _mic and C _p explained higher per cent variability in the SYI and yield of the crops. Results suggest that because of low cost and ease of estimation and also for upkeeping environmental conditions, C _frac1 may be used as a good indicator for assessment of soil as to its crop productivity. Responsible Editor: Hans Lambers.     

Arrhythmogenicity of radiofrequency ablation: Symptomatic non-sustained ventricular tachycardia and frequent premature ventricular ectopics after accessory pathway ablation

Radiofrequency ablation (RFA) has emerged as the preferred treatment modality with high success rate in cases with WPW syndrome. Arrhythmogenic complications are rarely reported after RFA, except for early or late recurrence of accessory pathway (AP) conduction. We present a unique case where the AP was successfully ablated, however, a new monomorphic PVC of similar morphology to the pre-excited beats developed within 30 min of RFA. She required medical management with sotalol to overcome her worsening symptom on follow-up. The ectopics resolved after 4 months.     

PARP1–TDP1 coupling for the repair of topoisomerase I–induced DNA damage

Poly(ADP-ribose) polymerases (PARP) attach poly(ADP-ribose) (PAR) chains to various proteins including themselves and chromatin. Topoisomerase I (Top1) regulates DNA supercoiling and is the target of camptothecin and indenoisoquinoline anticancer drugs, as it forms Top1 cleavage complexes (Top1cc) that are trapped by the drugs. Endogenous and carcinogenic DNA lesions can also trap Top1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1), a key repair enzyme for trapped Top1cc, hydrolyzes the phosphodiester bond between the DNA 3′-end and the Top1 tyrosyl moiety. Alternative repair pathways for Top1cc involve endonuclease cleavage. However, it is unknown what determines the choice between TDP1 and the endonuclease repair pathways. Here we show that PARP1 plays a critical role in this process. By generating TDP1 and PARP1 double-knockout lymphoma chicken DT40 cells, we demonstrate that TDP1 and PARP1 are epistatic for the repair of Top1cc. The N-terminal domain of TDP1 directly binds the C-terminal domain of PARP1, and TDP1 is PARylated by PARP1. PARylation stabilizes TDP1 together with SUMOylation of TDP1. TDP1 PARylation enhances its recruitment to DNA damage sites without interfering with TDP1 catalytic activity. TDP1–PARP1 complexes, in turn recruit X-ray repair cross-complementing protein 1 (XRCC1). This work identifies PARP1 as a key component driving the repair of trapped Top1cc by TDP1.     

Beneficial Effects of Simulated Gastro-Intestinal Digests of Fried Egg and Its Fractions on Blood Pressure,Plasma Lipids and Oxidative Stress in Spontaneously Hypertensive Rats

Background

We have previously characterized several antihypertensive peptides in simulated digests of cooked eggs and showed blood pressure lowering property of fried whole egg digest. However, the long-term effects of this hydrolysate and its fractions on blood pressure are not known. Therefore, the objectives of the study were to determine the effects of long term administration of fried whole egg hydrolysate and its fractions (i.e. egg white and egg yolk) on regulation of blood pressure and associated factors in cardiovascular disease such as plasma lipid profile and tissue oxidative stress.

Methods and Results

We used spontaneously hypertensive rats (SHR), an animal model of essential hypertension. Hydrolysates of fried egg and its fractions were prepared by simulated gastro-intestinal digestion with pepsin and pancreatin. 16–17 week old male SHRs were orally administered fried whole egg hydrolysate, non-hydrolyzed fried whole egg, egg white hydrolysate or egg yolk hydrolysates (either defatted, or not) daily for 18 days. Blood pressure (BP) and heart rate were monitored by telemetry. Animals were sacrificed at the end of the treatment for vascular function studies and evaluating plasma lipid profile and tissue oxidative stress. BP was reduced by feeding fried whole egg hydrolysate but not by the non-hydrolyzed product suggesting a critical role for in vitro digestion in releasing anti-hypertensive peptides. Egg white hydrolysate and defatted egg yolk hydrolysate (but not egg yolk hydrolysate) also had similar effects. Reduction in BP was accompanied by the restoration of nitric oxide (NO) dependent vasorelaxation and reduction of plasma angiotensin II. Fried whole egg hydrolysate also reduced plasma levels of triglyceride although it was increased by the non-hydrolyzed sample. Additionally the hydrolyzed preparations attenuated tissue oxidative stress.

Conclusion

Our results demonstrate that fried egg hydrolysates exert anti-hypertensive effects, improve plasma lipid profile and attenuate tissue oxidative stress in vivo.     

A Truncated Form of Rod Photoreceptor PDE6 β-Subunit Causes Autosomal Dominant Congenital Stationary Night Blindness by Interfering with the Inhibitory Activity of the γ-Subunit

Autosomal dominant congenital stationary night blindness (adCSNB) is caused by mutations in three genes of the rod phototransduction cascade, rhodopsin (RHO), transducin α-subunit (GNAT1), and cGMP phosphodiesterase type 6 β-subunit (PDE6B). In most cases, the constitutive activation of the phototransduction cascade is a prerequisite to cause adCSNB. The unique adCSNB-associated PDE6B mutation found in the Rambusch pedigree, the substitution p.His258Asn, leads to rod photoreceptors desensitization. Here, we report a three-generation French family with adCSNB harboring a novel PDE6B mutation, the duplication, c.928-9_940dup resulting in a tyrosine to cysteine substitution at codon 314, a frameshift, and a premature termination (p.Tyr314Cysfs*50). To understand the mechanism of the PDE6β1-314fs*50 mutant, we examined the properties of its PDE6-specific portion, PDE6β1-313. We found that PDE6β1-313 maintains the ability to bind noncatalytic cGMP and the inhibitory γ-subunit (Pγ), and interferes with the inhibition of normal PDE6αβ catalytic subunits by Pγ. Moreover, both truncated forms of the PDE6β protein, PDE6β1-313 and PDE6β1-314fs*50 expressed in rods of transgenic X. laevis are targeted to the phototransduction compartment. We hypothesize that in affected family members the p.Tyr314Cysfs*50 change results in the production of the truncated protein, which binds Pγ and causes constitutive activation of the phototransduction thus leading to the absence of rod adaptation.     

Identification of a novel set of scaffolding residues that are instrumental for the inhibitory property of Kunitz (STI) inhibitors

For canonical serine protease inhibitors (SPIs), scaffolding spacer residue Asn or Arg religates cleaved scissile peptide bond to offer efficient inhibition. However, several designed “mini‐proteins,” containing the inhibitory loop and the spacer(s) with trimmed scaffold behave like substrates, indicating that scaffolding region beyond the spacer is also important in the inhibitory process. To understand the loop‐scaffold compatibility, we prepared three chimeric proteins ECI^L‐WCI^S, ETI^L‐WCI^S, and STI^L‐WCI^S, where the inhibitory loop of ECI, ETI, and STI is placed on the scaffold of their homolog WCI. Results show that although ECI^L‐WCI^S and STI^L‐WCI^S behave like good inhibitors, ETI^L‐WCI^S behaves like a substrate. That means a set of loop residues (SRLRSAFI), offering strong trypsin inhibition in ETI, act as a substrate when they seat on the scaffold of WCI. Crystal structure of ETI^L‐WCI^S shows that the inhibitory loop is of noncanonical conformation. We identified three novel scaffolding residues Trp88, Arg74, and Tyr113 in ETI that act as barrier to confine the inhibitory loop to canonical conformation. Absence of this barrier in the scaffold of WCI makes the inhibitory loop flexible in ETI^L‐WCI^S leading to a loss of canonical conformation, explaining its substrate‐like behavior. Incorporation of this barrier back in ETI^L‐WCI^S through mutations increases its inhibitory power, supporting our proposition. Our study provides structural evidence for the contribution of remote scaffolding residues in the inhibitory process of canonical SPIs. Additionally, we rationalize why the loop‐scaffold swapping is not permitted even among the members of highly homologous inhibitors, which might be important in the light of inhibitor design.     

Design, synthesis, and subtype selectivity of 3,6-disubstituted β-carbolines at Bz/GABA(A)ergic receptors. SAR and studies directed toward agents for treatment of alcohol abuse

A series of 3,6-disubstituted β-carbolines was synthesized and evaluated for their in vitro affinities at α(x)β(3)γ(2) GABA(A)/benzodiazepine receptor subtypes by radioligand binding assays in search of α(1) subtype selective ligands to treat alcohol abuse. Analogues of β-carboline-3-carboxylate-t-butyl ester (βCCt, 1) were synthesized via a CDI-mediated process and the related 6-substituted β-carboline-3-carboxylates 6 including WYS8 (7) were synthesized via a Sonogashira or Stille coupling processes from 6-iodo-βCCt (5). The bivalent ligands of βCCt (32 and 33) were also designed and prepared via a palladium-catalyzed homocoupling process to expand the structure-activity relationships (SAR) to larger ligands. Based on the pharmacophore/receptor model, a preliminary SAR study on 34 analogues illustrated that large substituents at position-6 of the β-carbolines were well tolerated. As expected, these groups are proposed to project into the extracellular domain (L(Di) region) of GABA(A)/Bz receptors (see 32 and 33). Moreover, substituents located at position-3 of the β-carboline nucleus exhibited a conserved stereo interaction in lipophilic pocket L(1), while N(2) presumably underwent a hydrogen bonding interaction with H(1). Three novel β-carboline ligands (βCCt, 3PBC and WYS8), which preferentially bound to α1 BzR subtypes permitted a comparison of the pharmacological efficacies with a range of classical BzR antagonists (flumazenil, ZK93426) from several different structural groups and indicated these β-carbolines were 'near GABA neutral antagonists'. Based on the SAR, the most potent (in vitro) α(1) selective ligand was the 6-substituted acetylenyl βCCt (WYS8, 7). Earlier both βCCt and 3PBC had been shown to reduce alcohol self-administration in alcohol preferring (P) and high alcohol drinking (HAD) rats but had little or no effect on sucrose self-administration.(1-3) Moreover, these two β-carbolines were orally active, and in addition, were anxiolytic in P rats but were only weakly anxiolytic in rodents. These data prompted the synthesis of the β-carbolines presented here.