Structures of pahayokolides A and B, cyclic peptides from a Lyngbya sp

The isolation and structure elucidation of two cyclic peptides, pahayokolides A (1) and B (2), is described. Structural features determined for these compounds include a pendent N-acetyl-N-methyl leucine, both E- and Z-dehydrobutyrines, a homophenylalanine, and an unusual polyhydroxy amino acid that is most likely of mixed polyketide synthase/nonribosomal peptide synthase origin. These peptides were purified from a new species of cyanobacteria of the genus Lyngbya, which was isolated from a periphyton mat from the Florida Everglades.     

Quantitation and characterization of glutathionyl haemoglobin as an oxidative stress marker in chronic renal failure by mass spectrometry

OBJECTIVES: Glutathionyl haemoglobin (GS-Hb) belonging to the class of glutathionylated proteins has been investigated as a possible marker of oxidative stress in different chronic diseases. The purpose of this study was to examine whether glutathionyl haemoglobin can serve as an oxidative stress marker in non-diabetic chronic renal failure patients on different renal replacement therapies (RRT) through its quantitation, and characterization of the specific binding site of glutathione in haemoglobin molecule by mass spectrometric analysis. DESIGN AND METHODS: The study group consisted of non-diabetic chronic renal failure patients on renal replacement therapy (RRT): hemodialysis (HD), continuous ambulatory peritoneal dialysis (CAPD) and renal allograft transplant (Txp) patients. Haemoglobin samples of these subjects were analyzed by liquid chromatography electrospray ionization mass spectrometry for GS-Hb quantitation. Characterization of GS-Hb was done by tandem mass spectrometry. Levels of erythrocyte glutathione (GSH) and lipid peroxidation (as thiobarbituric acid reacting substances) were measured spectrophotometrically, while glycated haemoglobin (HbA1c) was measured by HPLC. RESULTS: GS-Hb levels were markedly elevated in the dialysis group and marginally in the transplant group as compared to the controls. GS-Hb levels correlated positively with lipid peroxidation and negatively with the erythrocyte glutathione levels in RRT groups indicating enhanced oxidative stress. De novo sequencing of the chymotryptic fragment of GS-Hb established that glutathione is attached to Cys-93 of the beta globin chain. Mass spectrometric quantitation of total glycated haemoglobin showed good agreement with HbA1c estimation by conventional HPLC method. CONCLUSIONS: Glutathionyl haemoglobin can serve as a clinical marker of oxidative stress in chronic debilitating therapies like RRT. Mass spectrometry provides a reliable analytical tool for quantitation and residue level characterization of different post-translational modifications of haemoglobin.     

The inflammation paradigm and coronary artery disease: What Celsus, Virchow and gene knock outs have taught us

Atherosclerotic vascular disease is a major cause of morbidity and mortality throughout the world. The clinical manifestations include coronary artery disease and myocardial infarction, cerebrovascular disease, renovascular disease and peripheral vascular disease. Initially considered a bland occlusive disease mediated to a great extent by lipids, atherosclerosis can now be considered an inflammatory disease, in its own right. This has led to a paradigm shift in disease management. We have come a long way since the time of Celsus, Galen, Virchow, Rokitansky and others when the components of the inflammatory cascade were first described. The development of mouse knock out models, improved molecular approaches to studying atheromatous blood vessels and development of sophisticated imaging and biomarker studies have enhanced our understanding of the molecular pathways in atherosclerosis. This brief review will attempt to weave together the historical, biochemical, immunological and molecular developments that have led to our current understanding of a deadly but treatable and potentially preventable disease.     

Nonclinical toxicology study of recombinant-plasmid DNA anti-rabies vaccines

The absence of standard guidelines from National and International regulatory agencies for the safety evaluation of biotechnology products challenges the ingenuity of toxicologists. At present, the development of standard pre-clinical toxicology protocols for such products is on an individual case basis. The present investigation is an attempt to evaluate the safety profile of the first indigenously developed DNA based anti-rabies vaccine in India. The test compounds were DNA rabies vaccine [DRV (100 microg)] and combination rabies vaccine (CRV (100 microg DRV and 1/50 dose of cell culture vaccine)), intended for clinical use by intramuscular route on 1, 7, 14 and 28 day. As per the regular mandatory requirements, the study has been designed to undertake acute (single dose--10 days), sub-chronic (repeat dose--28 days) and chronic (intended clinical dose--120 days) toxicity tests using three dose levels viz. therapeutic, average (2 x therapeutic dose) and highest dose (10 x therapeutic dose) exposure in Swiss Albino mice. The selection of the rodent model viz. Swiss Albino mice is based on affinity and rapid higher antibody response during the efficacy studies. Apart from physical, physiological, clinical, hematological and histopathology profiles of all target organs, the tier-I immunotoxicity parameters have also been monitored. There were no observational adverse effects even at levels of 10x therapeutic dose administration of DRV and CRV. The procedure also emphasizes on the designing of protocols for the products developed by recombinant technique.     

Coincidental loss of DOCK8 function in NLRP10-deficient and C3H/HeJ mice results in defective dendritic cell migration

Dendritic cells (DCs) are the primary leukocytes responsible for priming T cells. To find and activate naïve T cells, DCs must migrate to lymph nodes, yet the cellular programs responsible for this key step remain unclear. DC migration to lymph nodes and the subsequent T-cell response are disrupted in a mouse we recently described lacking the NOD-like receptor NLRP10 (NLR family, pyrin domain containing 10); however, the mechanism by which this pattern recognition receptor governs DC migration remained unknown. Using a proteomic approach, we discovered that DCs from Nlrp10 knockout mice lack the guanine nucleotide exchange factor DOCK8 (dedicator of cytokinesis 8), which regulates cytoskeleton dynamics in multiple leukocyte populations; in humans, loss-of-function mutations in Dock8 result in severe immunodeficiency. Surprisingly, Nlrp10 knockout mice crossed to other backgrounds had normal DOCK8 expression. This suggested that the original Nlrp10 knockout strain harbored an unexpected mutation in Dock8, which was confirmed using whole-exome sequencing. Consistent with our original report, NLRP3 inflammasome activation remained unaltered in NLRP10-deficient DCs even after restoring DOCK8 function; however, these DCs recovered the ability to migrate. Isolated loss of DOCK8 via targeted deletion confirmed its absolute requirement for DC migration. Because mutations in Dock genes have been discovered in other mouse lines, we analyzed the diversity of Dock8 across different murine strains and found that C3H/HeJ mice also harbor a Dock8 mutation that partially impairs DC migration. We conclude that DOCK8 is an important regulator of DC migration during an immune response and is prone to mutations that disrupt its crucial function.Dendritic cells (DCs) are crucial for the initiation of an adaptive immune response. Upon acquiring antigens in the periphery, DCs undergo a maturation process that includes antigen processing, cytokine production, and up-regulation of costimulatory molecules. A mature DC must then migrate from peripheral tissues to draining lymph nodes (LNs) to fulfill its role as an antigen-presenting cell that primes naïve T cells (1). Although the signals that induce this maturation process are now well-established (1), relatively little is understood about DC migration aside from the primary chemotactic cue provided by CCR7 that guides DCs to the LN (2, 3).We recently described a genetically modified NLRP10 (NLR family, pyrin domain containing 10) knockout strain in which this migration step was disrupted while leaving the remainder of the DC maturation program, including CCR7 expression, intact (4). NLRP10 is the only NOD-like receptor (NLR) without a leucine-rich repeat domain, the putative pathogen-associated molecular pattern (PAMP)–binding domain. It has been proposed to both positively and negatively regulate other NLRs, such as NOD1 and NLRP3, respectively (5, 6). Although we found that NLRP3 inflammasome activation was unaltered in the absence of NLRP10, we discovered that Nlrp10^−/− mice could not mount a productive T- or B-cell immune response due to a DC-intrinsic failure to emigrate out of inflamed tissues (4, 7).To understand the mechanism by which NLRP10 governs DC migration, we used an expression proteomic approach to identify molecules with altered expression in DCs generated from the Nlrp10^−/− strain and discovered a profound reduction in DOCK8 (dedicator of cytokinesis 8). DOCK8 is a guanine nucleotide exchange factor (GEF) that has two functional domains, DOCK homology region (DHR) 1 and DHR2 (8). In murine DCs, the DHR2 domain has been implicated in regulating the Rho GTPase CDC42 (cell division control protein 42 homolog), which in turn maintains cell polarity of mature DCs during migration (9, 10). Furthermore, mice harboring inactivating mutations in Dock8 lack marginal zone B-cell development, long-term antibody production following immunization, and memory CD8⁺ T-cell responses to viral infections (11, 12). In humans, inactivating mutations in Dock8 were recently identified as the primary genetic cause underlying autosomal recessive hyper-IgE syndrome (13). This syndrome presents with eczema, recurrent infections of the skin and respiratory tract, increased serum IgE, eosinophilia, recurrent fungal and viral infections, extensive food and environmental allergies, and, in certain patients, squamous cell dysplasia and carcinomas (14).Given that DOCK8 regulates a wide array of immunologic processes in mouse and human, we sought to understand how NLRP10 regulates DOCK8. To our surprise, we discovered that loss of DOCK8 in the Nlrp10^−/− strain was secondary to a point mutation within the Dock8 gene itself. In this study, we demonstrate that restoring DOCK8 function in the Nlrp10^−/− strain leads to normal DC migration in vivo. We further show that deletion of Dock8, as well as spontaneous mutation of Dock8 in another inbred strain of mice, results in defective DC migration and, depending on the degree of impaired migration, also abrogates CD4⁺ T-cell activation.     

Percutaneous closure of left ventricular-to-right atrial fistula after prosthetic mitral valve rereplacement using the Amplatzer duct occluder.

A 70-year-old female with a history of rheumatic heart disease underwent rereplacement of mitral valve mechanical prosthesis in May 2003. Seven months later, she presented with progressive exertional dyspnea, exercise intolerance, and a new holosystolic/diastolic murmur. Echocardiography confirmed a large shunt through a fistula in the inferior limbus of the atrial septum with left ventricular-to-right atrial communication. We report the novel use of the Amplatzer duct occluder for closure of the fistulous tract.     

Calibration and optimization of 3D digital breast tomosynthesis guided near infrared spectral tomography

Calibration of a three-dimensional multimodal digital breast tomosynthesis (DBT) x-ray and non-fiber based near infrared spectral tomography (NIRST) system is challenging but essential for clinical studies. Phantom imaging results yielded linear contrast recovery of total hemoglobin (HbT) concentration for cylindrical inclusions of 15 mm, 10 mm and 7 mm with a 3.5% decrease in the HbT estimate for each 1 cm increase in inclusion depth. A clinical exam of a patient’s breast containing both benign and malignant lesions was successfully imaged, with greater HbT was found in the malignancy relative to the benign abnormality and fibroglandular regions (11 μM vs. 9.5 μM). Tools developed improved imaging system characterization and optimization of signal quality, which will ultimately improve patient selection and subsequent clinical trial results.OCIS codes: (120.3890) Medical optics instrumentation, (170.0110) Imaging systems, (170.3830) Mammography