M. mazei glutamine synthetase and glutamine synthetase-GlnK1 structures reveal enzyme regulation by oligomer modulation.

Glutamine synthetases (GS) play central roles in cellular nitrogen assimilation. Although GS active-site formation requires the oligomerization of just two GS subunits, all GS form large, multi-oligomeric machines. Here we describe a structural dissection of the archaeal Methanosarcina mazei (Mm) GS and its regulation. We show that Mm GS forms unstable dodecamers. Strikingly, we show this Mm GS oligomerization property is leveraged for a unique mode of regulation whereby labile Mm GS hexamers are stabilized by binding the nitrogen regulatory protein, GlnK1. Our GS-GlnK1 structure shows that GlnK1 functions as molecular glue to affix GS hexamers together, stabilizing formation of GS active-sites. These data, therefore, reveal the structural basis for a unique form of enzyme regulation by oligomer modulation.

15-Deoxyspergualin hinders physical interaction between basic residues of transit peptide in PfENR and Hsp70-1.

The apicoplast of Plasmodium harbors several metabolic pathways. The enzymes required to perform these reactions are all nuclearly encoded and apicoplast targeted (NEAT) proteins. Plasmodium falciparum Enoyl-ACP Reductase (PfENR) is one such NEAT protein. The NEAT proteins have a transit peptide which is required for crossing the membranes of apicoplast. We studied the importance of basic residues like Arginine and Lysine within the transit peptide. Previous studies have suggested that all basic residues are essential for apicoplast trafficking. In this study, we demonstrate that only some of these residues are essential (K44, R48, K51, and R52), whereas others are dispensable (R40, K42, and K49). On mutating these specific residues, PfENR is not imported into the apicoplast and is mislocalized to the cytoplasm. We also demonstrate that these residues are also crucial for interaction with Hsp70-1, implying that interactions of Lysine 44, Arginine 48, Lysine 51, and Arginine 52 of the transit peptide with PfHsp70-1 are required for apicoplast trafficking. 15-Deoxyspergualin, which has earlier been proposed to interact with EEVD motif of PfHsp70-1 hinders the physical interaction between these cationic residues of PfENR and Hsp70-1. Hence, we propose that in the transport competent state of NEAT proteins some specific positively charged amino acids in the transit peptide interact with PfHsp70-1, and this interaction is essential for apicoplast targeting.

Relative adrenal insufficiency in cirrhotic patients with ascites (hepatoadrenal syndrome).

AIM: Relative adrenal insufficiency (RAI) has been reported in critically ill patients with cirrhosis. We evaluated the prevalence of RAI and its relationship to clinical course in non-septic cirrhosis patients with ascites. METHODS: The study included 66 consecutive non-septic cirrhosis patients with ascites. RAI was defined by a delta cortisol lower than 9 µg/dL and/or a peak cortisol lower than 18 µg/dL. RESULTS: Sixty-six patients with cirrhosis and ascites were studied. The mean Child-Turcotte-Pugh (CTP) and model for end stage liver disease (MELD) scores were 10.6 ±â€¯1.9 and 21.5 ±â€¯7.3, respectively. The prevalence of RAI in patients with cirrhosis and ascites was 47% (31/66). The prevalence of RAI in patients with and without spontaneous bacterial peritonitis, renal failure and type 1 hepatorenal syndrome (HRS) was comparable. Baseline hyponatremia was common in RAI (42% versus 17%, p = 0.026). There was a significant correlation of prevalence of RAI with prothrombin time, international normalized ratio, MELD scores and CTP class. During follow-up, there was no association between RAI and the risk to develop new infections, severe sepsis, type 1 HRS and death. CONCLUSIONS: RAI is common in non-septic cirrhotic patients with ascites and its prevalence increases with severity of liver disease. However, it does not affect the short-term outcome in these patients.

Bone morphogenetic protein-7 (BMP-7) augments insulin sensitivity in mice with type II diabetes mellitus by potentiating PI3K/AKT pathway.

A direct link between development of insulin resistance and the presence of chronic inflammation, in case of obesity exists, with cytokines playing an important role in glucose metabolism. Members of TGF-ß superfamily, including bone morphogenetic proteins (BMPs), have been shown to be involved in islet morphogenesis, establishment of ß-cell mass and adipose cell fate determination. Here, we demonstrate a novel and direct role of BMP-4 and -7 in the regulation of glucose homeostasis and insulin resistance. An age-dependent increase in serum BMP-4 and decrease in serum BMP-7 levels was observed in animal models of type II diabetes. In this study, BMP-7 and -4 have been demonstrated to have antagonistic effects on insulin signaling and thereby on glucose homeostasis. BMP-7 augmented glucose uptake in the insulin sensitive tissues such as the adipose and muscle by increasing Glut4 translocation to the plasma membrane through phosphorylation and activation of PDK1 and Akt, and phosphorylation and translocation of FoxO1 to the cytoplasm in liver/HepG2 cells. Restoration of BMP-7 levels in serum of diabetic animals resulted in decreased blood glucose levels in contrast to age matched untreated control groups, opening up a new therapeutic avenue for diabetes. On the contrary, BMP-4 inhibited insulin signaling through activation of PKC-θ isoform, and resulted in insulin resistance through the attenuation of insulin signaling. BMP-7 therefore is an attractive candidate for tackling a multifaceted disease such as diabetes, since it not only reduces body fat, but also strengthens insulin signaling, causing improved glucose uptake and ameliorating peripheral insulin resistance. © 2017 BioFactors, 43(2):195-209, 2017.

Impact and safety of endoscopic ultrasound guided fine needle aspiration on patients with cirrhosis and pyrexia of unknown origin in India.

BACKGROUND AND AIMS: Etiologic diagnosis of pyrexia of unknown origin is important in patients with cirrhosis for optimal management and to prevent flare up of infectious disease after liver transplantation. However, there is very limited literature available on this subject. The present study aimed to examine the safety and impact of endoscopic ultrasound (EUS) guided fine needle aspiration (FNA) in patients with cirrhosis. METHODS: The study was conducted between January 2014 and January 2016 at a tertiary care center. A total of 50 (47 lymph nodes, 3 adrenal) EUS guided FNAs were performed in 46 patients. Data are presented as median (25 - 75 IQR). RESULTS: The study included 46 patients (40 males) whose mean age was 47.9 ±â€Š11.1 (SD) years; mean Child-Turcotte-Pugh (CTP) score and mean MELD (Model for End-Stage Liver Disease) score were 10 (8 - 11) and 18 (12 - 20), respectively. The Child Pugh class was A in 4, B in 14, and C in 28 (including three patients with adrenal FNAs). Indications for FNA were pyrexia of unknown origin and lymphadenopathy on CT imaging. The cytopathological diagnoses were metastatic disease in 1 (adrenal), granulomatous change in 10 (6 positive with acid fast bacilli stain), histoplasmosis in three (two adrenals, one lymph node), 32 lymph nodes were reactive and four lymph node FNAs showed inadequate cellularity. The pathologic nodes had significantly lower long-to-short axis ratio [1.25 (1.09 - 1.28) versus 1.46 (1.22 - 1.87), P = 0.020]; a higher proportion of hypoechoic echotexture (5 versus 3, P = 0.017), and sharply defined borders (4 versus 2, P = 0.029). Complications included mild hepatic encephalopathy related to sedation in two patients with Child's C status. CONCLUSION: EUS guided FNA is safe in patients with cirrhosis and modified the management in 14/46 (30.4 %) patients.

Endoscopic ultrasound characteristics of tubercular lymphadenopathy in comparison to reactive lymph nodes.

AIM: Tuberculosis is a common disease in India with significant morbidity and mortality. Limited data is available on the description of tubercular lymphadenopathy on endoscopic ultrasound. METHODS: Retrospective data of 116 lymph nodes in 113 patients was evaluated at a tertiary care center. Lymphadenopathy in the mediastinum and abdomen were included. The study was aimed at identifying the endoscopic ultrasound (EUS) features of tubercular lymphadenopathy and comparing them with reactive lymphadenopathy in patients with pyrexia of unknown origin. RESULTS: The following features were suggestive of tubercular lymphadenopathy (n = 55) as compared to reactive lymphadenopathy (n = 61): hypoechoic echotexture (94.5% vs. 75.4%, p 0.004), patchy anechoic/hypoechoic areas (30.2% vs. 0%, p = 0.000), calcification (24.5% vs. 0%, p = 0.000), sharply demarcated borders (34.5% vs. 9.8%, p = 0.001), pus like material on aspirate (18.2% vs. 0%, p 0.000), and conglomeration of lymph nodes (10.9% vs. 0%, p = 0.009). The tubercular lymph nodes were significantly larger than reactive nodes at long axis and short axis diameter (2.4 ± 1.1 vs. 1.6 ± 0.6 cm, p < 0.001 and 1.5 ± 0.7 vs. 0.9 ± 0.3 cm, p = 0.001 respectively). On cytopathological examination, presence of necrosis (92.7% vs. 0%, p = 0.000) and granulomas (78.1% vs. 0%, p = 0.000) favored tubercular as compared to reactive lymphadenopathy. CONCLUSION: EUS features like hypoechoic echotexture, patchy anechoic/hypoechoic areas, calcification, sharply demarcated borders, conglomeration, purulent aspirate, larger size, and cytopathological presence of necrosis/granulomas are suggestive of tubercular as compared to reactive lymphadenopathy.

Endoscopic management of hilar biliary strictures.

Hilar biliary strictures are caused by various benign and malignant conditions. It is difficult to differentiate benign and malignant strictures. Postcholecystectomy benign biliary strictures are frequently encountered. Endoscopic management of these strictures is challenging. An endoscopic method has been advocated that involves placement of increasing number of stents at regular intervals to resolve the stricture. Malignant hilar strictures are mostly unresectable at the time of diagnosis and only palliation is possible.Endoscopic palliation is preferred over surgery or radiological intervention. Magnetic resonance cholangiopancreaticography is quite important in the management of these strictures. Metal stents are superior to plastic stents. The opinion is divided over the issue of unilateral or bilateral stenting.Minimal contrast or no contrast technique has been advocated during endoscopic retrograde cholangiopancreatography of these patients. The role of intraluminal brachytherapy, intraductal ablation devices, photodynamic therapy, and endoscopic ultrasound still remains to be defined.

Homocysteine alters the osteoprotegerin/RANKL system in the osteoblast to promote bone loss: pivotal role of the redox regulator forkhead O1.

In this study we determined the molecular mechanisms of how homocysteine differentially affects receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) synthesis in the bone. The results showed that oxidative stress induced by homocysteine deranges insulin-sensitive FOXO1 and MAP kinase signaling cascades to decrease OPG and increase RANKL synthesis in osteoblast cultures. We observed that downregulation of insulin/FOXO1 and p38 MAP kinase signaling mechanisms due to phosphorylation of protein phosphatase 2A (PP2A) was the key event that inhibited OPG synthesis in homocysteine-treated osteoblast cultures. siRNA knockdown experiments confirmed that FOXO1 is integral to OPG and p38 synthesis. Conversely homocysteine increased RANKL synthesis in osteoblasts through c-Jun/JNK MAP kinase signaling mechanisms independent of FOXO1. In the rat bone milieu, high-methionine diet-induced hyperhomocysteinemia lowered FOXO1 and OPG expression and increased synthesis of proresorptive and inflammatory cytokines such as RANKL, M-CSF, IL-1α, IL-1ß, G-CSF, GM-CSF, MIP-1α, IFN-γ, IL-17, and TNF-α. Such pathophysiological conditions were exacerbated by ovariectomy. Lowering the serum homocysteine level by a simultaneous supplementation with N-acetylcysteine improved OPG and FOXO1 expression and partially antagonized RANKL and proresorptive cytokine synthesis in the bone milieu. These results emphasize that hyperhomocysteinemia alters the redox regulatory mechanism in the osteoblast by activating PP2A and deranging FOXO1 and MAPK signaling cascades, eventually shifting the OPG:RANKL ratio toward increased osteoclast activity and decreased bone quality.

Crystallographic study of novel transthyretin ligands exhibiting negative-cooperativity between two thyroxine binding sites.

BACKGROUND: Transthyretin (TTR) is a homotetrameric serum and cerebrospinal fluid protein that transports thyroxine (T4) and retinol by binding to retinol binding protein. Rate-limiting tetramer dissociation and rapid monomer misfolding and disassembly of TTR lead to amyloid fibril formation in different tissues causing various amyloid diseases. Based on the current understanding of the pathogenesis of TTR amyloidosis, it is considered that the inhibition of amyloid fibril formation by stabilization of TTR in native tetrameric form is a viable approach for the treatment of TTR amyloidosis. METHODOLOGY AND PRINCIPAL FINDINGS: We have examined interactions of the wtTTR with a series of compounds containing various substitutions at biphenyl ether skeleton and a novel compound, previously evaluated for binding and inhibiting tetramer dissociation, by x-ray crystallographic approach. High resolution crystal structures of five ligands in complex with wtTTR provided snapshots of negatively cooperative binding of ligands in two T4 binding sites besides characterizing their binding orientations, conformations, and interactions with binding site residues. In all complexes, the ligand has better fit and more potent interactions in first T4 site i.e. (AC site) than the second T4 site (BD site). Together, these results suggest that AC site is a preferred ligand binding site and retention of ordered water molecules between the dimer interfaces further stabilizes the tetramer by bridging a hydrogen bond interaction between Ser117 and its symmetric copy. CONCLUSION: Novel biphenyl ether based compounds exhibit negative-cooperativity while binding to two T4 sites which suggests that binding of only single ligand molecule is sufficient to inhibit the TTR tetramer dissociation.