C-Terminus of the B-Chain of Relaxin-3 Is Important for Receptor Activity

Human relaxin-3 is a neuropeptide that is structurally similar to human insulin with two chains (A and B) connected by three disulfide bonds. It is expressed primarily in the brain and has modulatory roles in stress and anxiety, feeding and metabolism, and arousal and behavioural activation. Structure-activity relationship studies have shown that relaxin-3 interacts with its cognate receptor RXFP3 primarily through its B-chain and that its A-chain does not have any functional role. In this study, we have investigated the effect of modification of the B-chain C-terminus on the binding and activity of the peptide. We have chemically synthesised and characterized H3 relaxin as C-termini acid (both A and B chains having free C-termini; native form) and amide forms (both chains’ C-termini were amidated). We have confirmed that the acid form of the peptide is more potent than its amide form at both RXFP3 and RXFP4 receptors. We further investigated the effects of amidation at the C-terminus of individual chains. We report here for the first time that amidation at the C-terminus of the B-chain of H3 relaxin leads to significant drop in the binding and activity of the peptide at RXFP3/RXFP4 receptors. However, modification of the A-chain C-terminus does not have any effect on the activity. We have confirmed using circular dichroism spectroscopy that there is no secondary structural change between the acid and amide form of the peptide, and it is likely that it is the local C-terminal carboxyl group orientation that is crucial for interacting with the receptors.     

Simultaneous Post-cysteine(S-Acm) Group Removal Quenching of Iodine and Isolation of Peptide by One Step Ether Precipitation

The S-acetamidomethyl (Acm) protecting group is widely used in the chemical synthesis of peptides that contain one or more disulfide bonds. Treatment of peptides containing S-Acm protecting group with iodine results in simultaneous removal of the sulfhydryl protecting group and disulfide formation. However, the excess iodine needs to be quenched or adsorbed as quickly as possible after completion of the disulfide bond formation in order to minimize side reactions that are often associated with the iodination step. We report a simple method for simultaneous post-cysteine (Acm) group removal quenching of iodination and isolation. Use of large volumes of diethyl ether for direct precipitation action of the oxidized peptide from the 90 or 95% aqueous acetic acid solution affords nearly quantitative recovery of largely iodine-free peptide ready for direct purification. It was successfully applied to the synthesis of various peptides including human insulin-like peptide 3 analogues. Although recovery yields were comparable to the traditionally used ascorbic acid quenching method, this new approach offers significant advantages such as more simple utility, minimal side reactions, and greater cost effectiveness.     

Dust-acoustic solitary waves in a four-component adiabatic magnetized dusty plasma

Theoretical investigation has been made on obliquely propagating dust-acoustic (DA) solitary waves (SWs) in a magnetized dusty plasma which consists of non-inertial adiabatic electron and ion fluids, and inertial negatively as well as positively charged adiabatic dust fluids. The reductive perturbation method has been employed to derive the Korteweg-de Vries equation which admits a solitary wave solution for small but finite amplitude limit. It has been shown that the basic features (speed, height, thickness, etc.) of such DA solitary structures are significantly modified by adiabaticity of plasma fluids, opposite polarity dust components, and the obliqueness of external magnetic field. The SWs have been changed from compressive to rarefactive depending on the value of μ (a parameter determining the number of positive dust present in this plasma model). The present investigation can be of relevance to the electrostatic solitary structures observed in various dusty plasma environments (viz. cometary tails, upper mesosphere, Jupiter’s magnetosphere, etc.).     

Cdc5L interacts with ATR and is required for the S‐phase cell‐cycle checkpoint

Cell division cycle 5‐like protein (Cdc5L) is a core component of the putative E3 ubiquitin ligase complex containing Prp19/Pso4, Plrg1 and Spf27. This complex has been shown to have a role in pre‐messenger RNA splicing from yeast to humans; however, more recent studies have described a function for this complex in the cellular response to DNA damage. Here, we show that Cdc5L interacts physically with the cell‐cycle checkpoint kinase ataxia‐telangiectasia and Rad3‐related (ATR). Depletion of Cdc5L by RNA‐mediated interference methods results in a defective S‐phase cell‐cycle checkpoint and cellular sensitivity in response to replication‐fork blocking agents. Furthermore, we show that Cdc5L is required for the activation of downstream effectors or mediators of ATR checkpoint function such as checkpoint kinase 1 (Chk1), cell cycle checkpoint protein Rad 17 (Rad17) and Fanconi anaemia complementation group D2 protein (FancD2). In addition, we have mapped the ATR‐binding region in Cdc5L and show that a deletion mutant that is unable to interact with ATR is defective in the rescue of the checkpoint deficiency in Cdc5L‐depleted cells. These findings show a new function for Cdc5L in the regulation of the ATR‐mediated cell‐cycle checkpoint in response to genotoxic agents.     

Chemical synthesis and biological evaluation of an antimicrobial peptide gonococcal growth inhibitor

Gonococcal growth inhibitor 1 (GGI-1) is a 44-residue peptide with potent anti-Legionella activity. It has been isolated from Staphylococcus haemolyticus but, to date, its chemical synthesis has not been reported. Acquisition of this peptide via this means would enable a more detailed examination of its antimicrobial properties. However, its synthesis represents a significant challenge because of two predicted “difficult sequences” within the peptide. Its successful solid-phase assembly is reported in this paper, and was accomplished by use of simple palliative measures including the introduction of a single pseudo-proline isostere in order to counteract on-resin aggregation. The peptide had moderate antimicrobial activity against Escherichia coli but was inactive against another Gram-negative bacterium and two Gram-positive bacteria (Bacillus species). It had significant haemolytic activity, with a H₅₀ (concentration of peptide that causes 50?% haemolysis) of 20 and 125?μM for two blood samples from different donors. An alternative therapeutic index to that proposed for GGI-1 in a recent publication is proposed.     

Double mutation of two homologous genes YL1 and YL2 results in a leaf yellowing phenotype in soybean [Glycine max (L.) Merr]

Plant Molecular Biology - Two homologous, chloroplast located CAAX proteases were identified to be functional redundancy in determining soybean leaf color, and they probably play essential roles in...     

Abnormal production of pro- and anti-inflammatory cytokines by lupus monocytes in response to apoptotic cells

Monocytes are a key component of the innate immune system involved in the regulation of the adaptive immune response. Previous studies have focused on apoptotic cell clearance abnormalities in systemic lupus erythematosus (SLE) monocytes. However, whether SLE monocytes might express unique patterns of cytokine secretion in response to apoptotic cells is still unknown. Here, we used monocytes from healthy controls and SLE patients to evaluate the production of TNF-α and TGF-β in response to apoptotic cells. Upon recognition of apoptotic material, monocytes from healthy controls showed prominent TGF-β secretion (mean ± SD: 824.6±144.3 pg/ml) and minimal TNF-α production (mean ± SD: 32.6±2.1 pg/ml). In contrast, monocytes from SLE patients had prominent TNF-α production (mean ± SD: 302.2±337.5 pg/ml) and diminished TGF-β secretion (mean ± SD: 685.9±615.9 pg/ml), a difference that was statistically significant compared to normal monocytes (p≤10⁻⁶ for TNF-α secretion, and p = 0.0031 for TGF-β, respectively). Interestingly, the unique cytokine response by SLE monocytes was independent of their phagocytic clearance efficiency, opsonizing autoantibodies and disease activity. We further showed that nucleic acids from apoptotic cells play important role in the induction of TNF-α by lupus monocytes. Together, these observations suggest that, in addition to potential clearance defects, monocytes from SLE patients have an abnormal balance in the secretion of anti- and pro-inflammatory cytokines in response to apoptotic cells. Since the abnormal cytokine response to apoptotic material in SLE is not related to disease activity and opsonizing autoantibodies, it is possible that this response might be an intrinsic property of lupus monocytes. The studies focus attention on toll-like receptors (TLRs) and their downstream pathways as mediators of this response.     

High-molecular-weight polyethylene glycol protects cardiac myocytes from hypoxia- and reoxygenation-induced cell death and preserves ventricular function

Apoptosis plays a significant role in maladaptive remodeling and ventricular dysfunction following ischemia-reperfusion injury. There is a critical need for novel approaches to inhibit apoptotic cell death following reperfusion, as this loss of cardiac myocytes can progressively lead to heart failure. We investigated the ability and signaling mechanisms of a high-molecular-weight polyethylene glycol-based copolymer, PEG 15-20, to protect cardiac myocytes from hypoxia-reoxygenation (H-R)-induced cell death and its efficacy in preserving ventricular function following extended hypothermic ischemia and warm reperfusion as relevant to cardiac transplantation. Pretreatment of neonatal rat ventricular myocytes with a 5% PEG solution led to a threefold decline in apoptosis after H-R relative to untreated controls. There was a similar decline in caspase-3 activity in conjunction with inhibition of cytochrome c release from the inner mitochondrial membrane. Treatment with PEG also reduced reactive oxygen species production after H-R, and sarcolemmal lipid-raft architecture was preserved, consistent with membrane stabilization. Cell survival signaling was upregulated after H-R with PEG, as demonstrated by increased phosphorylation of Akt, GSK-3β, and ERK1/2. There was also maintenance of cardiac myocyte β-adrenergic signaling, which is critical for myocardial function. PEG 15-20 was very effective in preserving left ventricular function following prolonged hypothermic ischemia and warm reperfusion. PEG 15-20 has a potent protective antiapoptotic effect in cardiac myocytes exposed to H-R injury and may represent a novel therapeutic strategy to decrease myocardial cell death and ventricular dysfunction at the time of reperfusion during acute coronary syndrome or following prolonged donor heart preservation.