Pharmacology of insect GABA receptors

A GABA-operated Cl^– channel that is bicuculline-insensitive is abundant in the nervous tissue of cockroach, in housefly head preparations and thorax/abdomen preparations, and in similar preparations from several insect species. Bicuculline-insensitive GABA-operated Cl^– channels, which are rare in vertebrates, possess sites of action of benzodiazepines, steroids and insecticides that are pharmacologically-distinct from corresponding sites on vertebrate GABA_A receptors. The pharmacological profile of the benzodiazepine-binding site linked to an insect CNS GABA-operated Cl^– channel resembles more closely that of vertebrate peripheral benzodiazepine-binding sites. Six pregnane steroids and certain polychlorocycloalkane insecticides, which are active att-butylbicy-clophosphorothionate (TBPS)-binding sites, also differ in their effectiveness on vertebrate and insect GABA receptors. Radioligand binding and physiological studies indicate that in insects there may be subtypes of the GABA receptor. Molecular biology offers experimental approaches to understanding the basis of this diversity.Special issue dedicated to Dr. Eugene Roberts     

cDNA and gene structure for a human subtilisin-like protease with cleavage specificity for paired basic amino acid residues.

A cDNA encoding the human fur gene product was isolated from a human hepatoma cell line. The cDNA encodes a protein with significant amino acid sequence identity to the prokaryotic subtilisin family of serine proteases. More extensive sequence identity was found when the protein was compared with eukaryotic proteases such as PRB1 of Saccharomyces cerevisiae, and with PC2 and PC3, the only other known mammalian subtilisin-like proteases. In contrast to these proteins, however, the fur gene product shares a more extensive topographic and functional homology with the KEX2 endoprotease of S. cerevisiae. Each protease contains a signal peptide, a glycosylated extra cytoplasmic domain, a hydrophobic membrane-spanning region, and a short, hydrophilic "tail" sequence. As with KEX2, the expressed human protease was shown to cleave mammalian proproteins at their paired basic amino acid processing sites. We have, therefore, proposed the function-based acronym PACE (paired basic amino acid cleaving enzyme) for this prototypic mammalian proprotein processing enzyme.     

Holding back the microfilament--structural insights into actin and the actin-monomer-binding proteins of apicomplexan parasites

Parasites from the phylum Apicomplexa are responsible for several major diseases of man, including malaria and toxoplasmosis. These highly motile protozoa use a conserved actomyosin-based mode of movement to power tissue traversal and host cell invasion. The mode termed as 'gliding motility' relies on the dynamic turnover of actin, whose polymerisation state is controlled by a markedly limited number of identifiable regulators when compared with other eukaryotic cells. Recent studies of apicomplexan actin regulator structure-in particular those of the core triad of monomer-binding proteins, actin-depolymerising factor/cofilin, cyclase-associated protein/Srv2, and profilin-have provided new insights into possible mechanisms of actin regulation in parasite cells, highlighting divergent structural features and functions to regulators from other cellular systems. Furthermore, the unusual nature of apicomplexan actin itself is increasingly coming into the spotlight. Here, we review recent advances in understanding of the structure and function of actin and its regulators in apicomplexan parasites. In particular we explore the paradox between there being an abundance of unpolymerised actin, its having a seemingly increased potential to form filaments relative to vertebrate actin, and the apparent lack of visible, stable filaments in parasite cells.     

The development, application and limitations of breast cancer cell lines to study tamoxifen and aromatase inhibitor resistance

Estrogen plays important roles in hormone receptor-positive breast cancer. Endocrine therapies, such as the antiestrogen tamoxifen, antagonize the binding of estrogen to estrogen receptor (ER), whereas aromatase inhibitors (AIs) directly inhibit the production of estrogen. Understanding the mechanisms of endocrine resistance and the ways in which we may better treat these types of resistance has been aided by the development of cellular models for resistant breast cancers. In this review, we will discuss what is known thus far regarding both de novo and acquired resistance to tamoxifen or AIs. Our laboratory has generated a collection of AI- and tamoxifen-resistant cell lines in order to comprehensively study the individual types of resistance mechanisms. Through the use of microarray analysis, we have determined that our cell lines resistant to a particular AI (anastrozole, letrozole, or exemestane) or tamoxifen are distinct from each other, indicating that these mechanisms can be quite complex. Furthermore, we will describe two novel de novo AI-resistant cell lines that were generated from our laboratory. Initial characterization of these cells reveals that they are distinct from our acquired AI-resistant cell models. In addition, we will review potential therapies which may be useful for overcoming resistant breast cancers through studies using endocrine resistant cell lines. Finally, we will discuss the benefits and shortcomings of cell models. Together, the information presented in this review will provide us a better understanding of acquired and de novo resistance to tamoxifen and AI therapies, the use of appropriate cell models to better study these types of breast cancer, which are valuable for identifying novel treatments and strategies for overcoming both tamoxifen and AI-resistant breast cancers.     

Gestational hypertension and the developmental origins of cardiac hypertrophy and diastolic dysfunction

The developmental origins of health and disease refer to the theory that adverse maternal environments influence fetal development and the risk of cardiovascular disease in adulthood. We used the chronically hypertensive atrial natriuretic peptide knockout (ANP?/?) mouse as a model of gestational hypertension, and attempted to determine the effect of gestational hypertension on left ventricular (LV) structure and function in adult offspring. We crossed normotensive ANP+/+ females with ANP?/? males (yielding ANP+/?^WT offspring) and hypertensive ANP?/? females with ANP+/+ males (yielding ANP+/?^KO offspring). Cardiac gene expression was measured using real-time quantitative PCR. Cardiac function was assessed using echocardiography. Daily injections of isoproterenol (ISO) were used to induce cardiac stress. Collagen deposition was assessed using picrosirius red staining. All mice were 10 weeks of age. Gestational hypertension resulted in significant LV hypertrophy in offspring, with no change in LV function. Treatment with ISO resulted in significant LV diastolic dysfunction with a restrictive filling pattern (increased E/A ratio and E/e′) and interstitial myocardial fibrosis only in ANP+/?^KO and not ANP+/?^WT offspring. Gestational hypertension programs adverse LV structural and functional remodeling in offspring. These data suggest that adverse maternal environments may increase the risk of heart failure in offspring later in life.     

Adverse Drug Reactions in Hospitalised Children in Germany Are Decreasing: Results of a Nine Year Cohort-Based Comparison

Background

In recent years, efforts have been made to improve paediatric drug therapy. The aim of this research was to investigate any changes regarding the frequency and nature of adverse drug reactions (ADRs) in hospitalized children in one paediatric general medical ward over a 9-year period.

Methodology

Two prospective observational cohort studies were conducted at a large University hospital in Germany in 1999 and 2008, respectively. Children aged 0–18 years admitted to the study ward during the study periods were included. ADRs were identified using intensive chart review. Uni- and multivariable regression has been used for data analysis.

Results

A total of 520 patients (574 admissions) were included [1999: n = 144 (167); 2008: n = 376 (407)]. Patients received a total of 2053 drugs [median 3, interquartile range (IQR) 2–5]. 19% of patients did not receive any medication. Median length of stay was 4 days (IQR 3–7; range 1–190 days) with a significantly longer length of stay in 1999. The overall ADR incidence was 13.1% (95% CI, 9.8–16.3) varying significantly between the two study cohorts [1999: 21.9%, 95% CI, 14.7–29.0; 2008: 9.2%, 95% CI, 5.9–12.5 (p<0.001)]. Antibacterials and corticosteroids for systemic use caused most of the ADRs in both cohorts (1999; 2008). Exposure to systemic antibacterials decreased from 62.9% to 43.5% whereas exposure to analgesics and anti-inflammatory drugs increased from 17.4% to 45.2%, respectively. The use of high risk drugs decreased from 75% to 62.2%. In 1999, 45.7% and in 2008 96.2% of ADRs were identified by treating clinicians (p<0.001).

Conclusions

Between 1999 and 2008, the incidence of ADRs decreased significantly. Improved treatment strategies and an increased awareness of ADRs by physicians are most likely to be the cause for this positive development. Nevertheless further research on ADRs particularly in primary care and the establishment of prospective pharmacovigilance systems are still needed.     

Polymorphic phases of galactocerebrosides: spectroscopic evidence of lamellar crystalline structures

Fourier transform infrared spectroscopy was applied to study the structural and thermal properties of bovine brain galactocerebroside (GalCer) containing amide linked non-hydroxylated or alpha-hydroxy fatty acids (NFA- and HFA-GalCer, respectively). Over the temperature range 0-90 degrees C, both GalCer displayed complex thermal transitions, characteristic of polymorphic phase behavior. Upon heating, aqueous dispersions of NFA- and HFA-GalCer exhibited high order-disorder transition temperatures near 80 and 72 degrees C, respectively. En route to the chain melting transition, the patterns of the amide I band of NFA-GalCer were indicative of two different lamellar crystalline phases, whereas those of HFA-GalCer were suggestive of lamellar gel and crystalline bilayers. Cooling from the liquid-crystalline phase resulted in the formation of another crystalline phase of NFA-GalCer and a gel phase of HFA-GalCer, with a phase transition near 62 and 66 degrees C, respectively. Prolonged incubation of GalCer bilayers at 38 degrees C revealed conversions among lamellar crystalline phases (NFA-GalCer) or between lamellar gel and crystalline bilayer structures (HFA-GalCer). Spectral changes indicated that the temperature and/or time induced formation of the lamellar crystalline structures of NFA- and HFA-GalCer was accompanied by partial dehydration and by rearrangements of the hydrogen bonding network and bilayer packing mode of GalCer.     

Enhanced binding of radioligands to receptors of γ-aminobutyric acid and benzodiazepine by a new anticonvulsive agent,LY81067

A diaryltriazine, LY81067, effectively protects against pentylenetetrazole- and picrotoxin-induced convulsions in mice, with ED₅₀ values of 5.7 and 5.8 mg/kg i.p., respectively. LY81067 enhances the binding of both ³H-GABA and ³H-flunitrazepam to specific sites in rat brain membranes. The degree of enhancement by LY81067 varies from one brain region to another and is different for the binding of ³H-GABA and ³H-flunitrazepam. In cortical membranes, LY81067 increases the affinity of ³H-GABA for both high and low affinity sites and increases the number of sites. LY81067 increases the affinity of ³H-flunitrazepam for its binding sites without greatly increasing the number of sites. Like the pyrazolopyridines, the enhancement of ³H-flunitrazepam binding by LY81067 is dependent on chloride or related anions and is reversed by picrotoxin, suggesting that LY81067 exerts its anticonvulsant effects by binding to or near picrotoxin binding sites. The differential effects of LY81067 on the enhancements of ³H-GABA and ³H-flunitrazepam binding in several brain regions suggest extensive multiplicity of GABA/benzodiazepine/picrotoxin/anioin receptor complexes.