New non-steroidal aromatase inhibitors: focus on R76713

R76713 is a novel triazole derivative which selectively blocks the cytochrome P450-dependent aromatase. In human placental microsomes, in FSH-stimulated rat and human granulosa cells and in human adipose stromal cells, 50% inhibition of estradiol biosynthesis was obtained at drug concentrations of 2-10 nM. In PMSG-injected female rats, R76713 lowered plasma estradiol levels by 50 and 90% 2 h after single oral doses of 0.005 and 0.05 mg/kg respectively. After 1 mg/kg, estradiol levels were suppressed by 90% for 16 h. In male cynomolgus monkeys, R76713 dose-dependently (0.03-10 micrograms/kg) inhibited peripheral aromatization with an ED50 of 0.13 microgram/kg without altering metabolic clearance rates and conversion ratios. In vitro R76713 had no effect on other P450-dependent steroidogenic enzymes up to 1000 nM at least. In rats, LHRH-, ACTH- and sodium-deprived diet stimulated plasma testosterone, corticosterone and aldosterone levels were not modified 2 h after single oral administrations of R76713 (up to 20 mg/kg). Furthermore, R76713 did not show any in vitro or in vivo estrogenic or antiestrogenic property. R76713 also induced regression of DMBA-induced mammary tumors after daily oral administration of 1 mg/kg b.i.d. In male volunteers (n = 4), a single oral dose of 5 and 10 mg lowered median plasma estradiol levels from 70 pM to the detection limit of the assay (40 pM) 4, 8 and 24 h after intake whereas no changes were detected after placebo administration. In premenopausal women (n = 15), receiving a single oral dose of 20 mg, median plasma estradiol levels decreased from 389 pM (before) to 168, 133 and 147 pM, 4, 8 and 24 h after intake whereas they remained above 420 pM after placebo (n = 7).     

New focus on regulation of the rod photoreceptor phosphodiesterase

Rod photoreceptor phosphodiesterase (PDE6) is the key catalytic enzyme of visual phototransduction. PDE6 is the only member of the phosphodiesterase family that consists of a heterodimeric catalytic core composed of PDE6α and PDE6β subunits and two inhibitory PDE6γ subunits. Both PDE6α and PDE6β contain two regulatory GAF domains and one catalytic domain. GAF domains and the tightly bound PDE6γ subunits allosterically regulate the activity of the catalytic domain in association with the GTP-bound transducin alpha subunit (G_tα-GTP). Recent cryo-electron microscopy structures of the PDE6αγβγ and PDE6αγβγ-(G_tα-GTP)₂ complexes have provided valuable knowledge shedding additional light on the allosteric activation of PDE6 by G_tα-GTP. Here we discuss recent developments in our understanding of the mechanism of PDE6 activation.     

New biological aspects of chromogranin A-derived peptides: focus on vasostatins

Chromogranin A (CgA), one component of the granin family, represents the major soluble protein co-stored and co-released with catecholamines, within chromaffin cells secretory granules. It is considered a diagnostic and prognostic marker of several diseases, including a variety of tumours and cardiac heart failure. It also represents a precursor of biologically active fragments, generated after proteolytic cleavage at the level of the multiple pairs of dibasic sites which enrich its sequence. CgA, and its derived fragments show an old evolutionary history being ubiquitously present throughout the animal word, from mammals to invertebrates. Their biological functions include control of hormone production, and several paracrine and autocrine actions mainly attributed to its derived peptides. Two N-terminal fragments, named vasostatins 1 (VS-1: CgA(1-76)) and vasostatin 2 (VS-2: CgA(1-113)) due to their ability to dilate pre-constricted vessels, exert a large spectrum of homeostatic actions, including antifungal and antimicrobial effect, modulation of cell adhesion, and inhibition of parathyroid hormone secretion. Recently, on isolated heart preparations from eel, frog and rat they were shown to act as negative inotropic agents able to counteract the effects of beta-adrenergic stimulation. This short note introduces the abstracts of the contributions at the "International Workshop on Vasostatins and Chromogranin A-derived peptides" (Island of Capri, Italy; September 2005). The Workshop was focused on recent findings on the role of vasostatins (VSs) in cardiovascular and gastrointestinal systems, extracellular fluids composition, and innate immunity. Particular attention has been given to the still elusive mechanism of action of these peptides.     

A focus on fixation

Three fixation issues related to immunostaining are discussed here: 1) Generally, a tissue block is fixed, then embedded and sectioned (pre-fixation). The type of fixative applied, crosslinking or coagulating, has an impact on selecting an epitope retrieval method. Individual antigens have a fixation–retrieval characteristic. 2) A long fixation time, especially with crosslinking fixatives, may compromise the result of immunostaining. This negative effect varies among different antigens and can be partially restored by applying a more sensitive/efficient detection system such as tyramide amplification. 3) Sections cut from a fresh frozen tissue block usually are acetone fixed (post-fixation). This was accepted as the “gold standard” for a long time. Post-fixation, however, may have serious consequences for preservation of small peptides leaking from the cut open cells, whereas this is not the case with pre-fixed intact cells. Consequently, the concept of an acetone post-fixed cryostat tissue section as “gold standard” no longer exists and a more appropriate use of the terms immunohistochemistry and immunocytochemistry therefore seems justified. For many antibodies, it is not known whether a formalin fixed, paraffin embedded tissue specimen is appropriate. Suggestions are made for creating a positive control cell block for testing such antibodies.     

A focus on fixation

Three fixation issues related to immunostaining are discussed here: 1) Generally, a tissue block is fixed, then embedded and sectioned (pre-fixation). The type of fixative applied, crosslinking or coagulating, has an impact on selecting an epitope retrieval method. Individual antigens have a fixation-retrieval characteristic. 2) A long fixation time, especially with crosslinking fixatives, may compromise the result of immunostaining. This negative effect varies among different antigens and can be partially restored by applying a more sensitive/efficient detection system such as tyramide amplification. 3) Sections cut from a fresh frozen tissue block usually are acetone fixed (post-fixation). This was accepted as the “gold standard” for a long time. Post-fixation, however, may have serious consequences for preservation of small peptides leaking from the cut open cells, whereas this is not the case with pre-fixed intact cells. Consequently, the concept of an acetone post-fixed cryostat tissue section as “gold standard” no longer exists and a more appropriate use of the terms immunohistochemistry and immunocytochemistry therefore seems justified. For many antibodies, it is not known whether a formalin fixed, paraffin embedded tissue specimen is appropriate. Suggestions are made for creating a positive control cell block for testing such antibodies.     

Shifting sands in science.

Pertussis is a severe epidemic disease that causes significant morbidity and mortality in unimmunized children. It is now clear, however, that adults with atypical disease account for many of the cases and are often responsible for transmission to susceptible infants. Because of the extent of unrecognized pertussis in the adult population, mass pediatric immunization has been successful in controlling the disease in children but not in reducing the presence of the organism in the United States. Pertussis immunization of children is associated with a high rate of side reactions and is temporally related to severe neurologic disease and death in infants. These events are often considered to be reactions, but available scientific evidence indicates that few, if any, are actually caused by pertussis immunization. Pertussis vaccine reactogenicity in adults is anecdotally considered to be worse than in children, but direct studies do not support that. In the context of current programs emphasizing adult immunization, consideration should be given to booster doses of diphtheria-tetanus-pertussis in adults, using component pertussis vaccines when they become available.     

New insights and perspectives on intrarenal renin-angiotensin system: focus on intracrine/intracellular angiotensin II

Although renin, the rate-limiting enzyme of the renin-angiotensin system (RAS), was first discovered by Robert Tigerstedt and Bergman more than a century ago, the research on the RAS still remains stronger than ever. The RAS, once considered to be an endocrine system, is now widely recognized as dual (circulating and local/tissue) or multiple hormonal systems (endocrine, paracrine and intracrine). In addition to the classical renin/angiotensin I-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II receptor (AT₁/AT₂) axis, the prorenin/(Pro)renin receptor (PRR)/MAP kinase axis, the ACE2/Ang (1-7)/Mas receptor axis, and the Ang IV/AT₄/insulin-regulated aminopeptidase (IRAP) axis have recently been discovered. Furthermore, the roles of the evolving RAS have been extended far beyond blood pressure control, aldosterone synthesis, and body fluid and electrolyte homeostasis. Indeed, novel actions and underlying signaling mechanisms for each member of the RAS in physiology and diseases are continuously uncovered. However, many challenges still remain in the RAS research field despite of more than one century's research effort. It is expected that the research on the expanded RAS will continue to play a prominent role in cardiovascular, renal and hypertension research. The purpose of this article is to review the progress recently being made in the RAS research, with special emphasis on the local RAS in the kidney and the newly discovered prorenin/PRR/MAP kinase axis, the ACE2/Ang (1-7)/Mas receptor axis, the Ang IV/AT₄/IRAP axis, and intracrine/intracellular Ang II. The improved knowledge of the expanded RAS will help us better understand how the classical renin/ACE/Ang II/AT₁ receptor axis, extracellular and/or intracellular origin, interacts with other novel RAS axes to regulate blood pressure and cardiovascular and kidney function in both physiological and diseased states.     

A European focus on proteomics

A report on the First International Symposium of the Austrian Proteomics Platform, Seefeld, Austria, 26-29 January 2004.