Mechanism of hypochlorous acid-mediated heme destruction and free iron release

Here, we show that hypochlorous acid (HOCl), a potent neutrophil-generated oxidant, can mediate destruction of free heme (Ht) and the heme precursor, protoporphyrin IX (PPIX). Ht displays a broad Soret absorbance peak centered at 365 and 394 nm, indicative of the presence of monomer and μ-oxo-dimer. Oxidation of Ht by HOCl was accompanied by a marked decrease in the Soret absorption peak and release of free iron. Kinetic measurements showed that the Ht-HOCl reaction was triphasic. The first two phases were HOCl concentration dependent and attributable to HOCl binding to the monomeric and dimeric forms. The third phase was HOCl concentration independent and attributed to Ht destruction with the release of free iron. HPLC and LC-ESI-MS analyses of the Ht-HOCl reaction revealed the formation of a number of degradation products, resulting from the cleavage or modification of one or more carbon-methene bridges of the porphyrin ring. Similar studies with PPIX showed that HOCl also mediated tetrapyrrole ring destruction. Collectively, this work demonstrates the ability of HOCl to modulate destruction of heme, through a process that occurs independent of the iron molecule that resides in the porphyrin center. This phenomenon may play a role in HOCl-mediated oxidative injury in pathological conditions.     

Targeted inhibition of hedgehog signaling by cyclopamine prodrugs for advanced prostate cancer

A promising agent for use in prostate cancer therapy is the Hedgehog (Hh) signaling pathway inhibitor, cyclopamine. This compound, however, has the potential for causing serious side effects in non-tumor tissues. To minimize these bystander toxicities, we have designed and synthesized two novel peptide-cyclopamine conjugates as prostate-specific antigen (PSA)-activated prodrugs for use against prostate cancer. These prodrugs were composed of cyclopamine coupled to one of two peptides (either HSSKLQ or SSKYQ) that can be selectively cleaved by PSA, converting the mature prodrug into an active Hedgehog inhibitor within the malignant cells. Of the two prodrugs, Mu-SSKYQ-Cyclopamine was rapidly hydrolyzed, with a half-life of 3.2h, upon incubation with the PSA enzyme. Thus, modulating cyclopamine at the secondary amine with PSA-cleavable peptides is a promising strategy for developing prodrugs to target prostate cancer.     

The advancement of telomere quantification methods

Molecular Biology Reports - Telomeres, guanine rich DNA sequences, which are found at both ends of human chromosomes, play a vital role in genome protection. These repetitive nucleotide sequences...     

A Lipid Transfer Protein Signaling Axis Exerts Dual Control of Cell-Cycle and Membrane Trafficking Systems

1. Download high-res image (77KB)
2. Download full-size image

     

Photoperiodic regulation of annual testicular events in the Indian major carp Catla catla

The importance of photoperiods in the regulation of annual testicular events in the carp Catla catla was evaluated by subjecting them to either long (16 h light : 8 h dark) or short (8 h light : 16 h dark) photoperiods for 30 days during the preparatory, prespawning, spawning and postspawning phases of an annual gonadal cycle. In each reproductive phase, testicular responsiveness to subjected photoperiods was determined by comparing the gonadal status in corresponding groups of control or natural photoperiodic fish. The values of testicular weight, gametogenic index, as well as testicular activity of two steroidogenic enzymes (Δ⁵3β‐, and 17β‐hydroxysteroid dehydrogenase), and the serum titre of testosterone were considered as the indices of functional status of the testis in the fish concerned. During the prespawning phase, exposure of fish to a daily long photoperiod schedule resulted in precocious maturation of testis, while retardation of testicular growth was noted under the influences of short photoperiod. However, none of the employed photo‐schedules could influence the gametogenic and steroidogenic functions of the testis in the remaining part of the gonadal cycle. Collectively, the present study provides evidence for the first time that in the case of a commercially important carp, Catla catla, artificial, long photoperiods may be used for advanced testicular maturation, while reductions in maturation‐associated growth and deterioration in flesh quality may be avoided by submitting the fish to shorter day lengths during the prespawning phase of the reproductive cycle.     

Human immunodeficiency virus rev protein recognizes a target sequence in rev-responsive element RNA within the context of RNA secondary structure.

Human immunodeficiency virus type 1 Rev protein modulates the distribution of viral mRNAs from the nucleus to the cytoplasm by interaction with a highly structured viral RNA sequence, the Rev-responsive element (RRE). To identify the minimal functional elements of RRE, we evaluated mutant RREs for Rev binding in vitro and Rev response in vivo in the context of a Gag expression plasmid. The critical functional elements fold into a structure composed of a stem-loop A, formed by the ends of the RRE, joined to a branched stem-loop B/B1/B2, between bases 49 and 113. The 5' 132 nucleotides of RRE, RREDDE, which possessed a similar structure, bound Rev efficiently but were nonfunctional in vivo, implying separate binding and functional domains within the RRE. Excision of stem-loop A reduced Rev binding significantly and abolished the in vivo Rev response. The B2 branch could be removed without severe impairment of binding, but deletions in the B1 branch significantly reduced binding and function. However, deletion of 12 nucleotides, including the 5' strand of stem B, abolished both binding and function, while excision of the 3' strand of stem B only reduced them. Maintenance of the native RRE secondary structure alone was not sufficient for Rev recognition. Many mutations that altered the primary structure of the critical region while preserving the original RNA conformation were Rev responsive. However, mutations that changed a 5'..CACUAUGGG..3' sequence in the B stem, without affecting the overall structure abolished both in vitro Rev binding and the in vivo Rev response.     

Synthesis and pharmacological characterization of functionalized 6-piperazin-1-yl-purines as cannabinoid receptor 1 (CB1) inverse agonists

Antagonists of peripheral type 1 cannabinoid receptors (CB1) may have utility in the treatment of obesity, liver disease, metabolic syndrome and dyslipidemias. We have targeted analogues of the purine inverse agonist otenabant (1) for this purpose. The non-tissue selective CB1 antagonist rimonabant (2) was approved as a weight-loss agent in Europe but produced centrally mediated adverse effects in some patients including dysphoria and suicidal ideation leading to its withdrawal. Efforts are now underway to produce compounds with limited brain exposure. While many structure-activity relationship (SAR) studies of 2 have been reported, along with peripheralized compounds, 1 remains relatively less studied. In this report, we pursued analogues of 1 in which the 4-aminopiperidine group was switched to piperazine group to enable a better understanding of SAR to eventually produce compounds with limited brain penetration. To access a binding pocket and modulate physical properties, the piperazine was functionalized with alkyl, heteroalkyl, aryl and heteroaryl groups using a variety of connectors, including amides, sulfonamides, carbamates and ureas. These studies resulted in compounds that are potent antagonists of hCB1 with high selectivity for hCB1 over hCB2. The SAR obtained led to the discovery of 65 (Ki?=?4?nM, >1,000-fold selective for hCB1 over hCB2), an orally bioavailable aryl urea with reduced brain penetration, and provides direction for discovering peripherally restricted compounds with good in vitro and in vivo properties.