Evolution of base composition in the insulin and insulin-like growth factor genes

The genomes of homeothermic (warm-blooded) vertebrates are mosaic interspersions of homogeneously GC-rich and GC-poor regions (isochores). Evolution of genome compartmentalization and GC-rich isochores is hypothesized to reflect either selective advantages of an elevated GC content or chromosome location and mutational pressure associated with the timing of DNA replication in germ cells. To address the present controversy regarding the origins and maintenance of isochores in homeothermic vertebrates, newly obtained as well as published nucleotide sequences of the insulin and insulin-like growth factor (IGF) genes, members of a well-characterized gene family believed to have evolved by repeated duplication and divergence, were utilized to examine the evolution of base composition in nonconstrained (flanking) and weakly constrained (introns and fourfold degenerate sites) regions. A phylogeny derived from amino acid sequences supports a common evolutionary history for the insulin/IGF family genes. In cold- blooded vertebrates, insulin and the IGFs were similar in base composition. In contrast, insulin and IGF-II demonstrate dramatic increases in GC richness in mammals, but no such trend occurred in IGF- I. Base composition of the coding portions of the insulin and IGF genes across vertebrates correlated (r = 0.90) with that of the introns and flanking regions. The GC content of homologous introns differed dramatically between insulin/IGF-II and IGF-I genes in mammals but was similar to the GC level of noncoding regions in neighboring genes. Our findings suggest that the base composition of introns and flanking regions is determined by chromosomal location and the mutational pressure of the isochore in which the sequences are embedded. An elevated GC content at codon third positions in the insulin and the IGF genes may reflect selective constraints on the usage of synonymous codons.      

High definition bronchoscopy: a randomized exploratory study of diagnostic value compared to standard white light bronchoscopy and autofluorescence bronchoscopy

Background

Videobronchoscopy is an essential diagnostic procedure for evaluation of the central airways and pivotal for the diagnosis and staging of lung cancer. Technological improvements have resulted in high definition (HD) images with advanced real time image enhancement techniques (i-scan).

Objectives

In this study we aimed to explore the sensitivity of HD+ i-scan bronchoscopy for detection of epithelial changes like vascular abnormalities and suspicious preinvasive lesions, and tumors.

Methods

In patients scheduled for a therapeutic or diagnostic procedure under general anesthesia videos of the bronchial tree were made using 5 videobronchoscopy modes in random order: normal white light videobronchoscopy (WLB), HD-bronchoscopy (HD), HD bronchoscopy with surface enhancement technique (i-scan1), HD with surface- and tone enhancement technique (i-scan2) and dual mode autofluorescence videobronchoscopy (AFB). The videos were scored in random order by two independent and blinded expert bronchoscopists.

Results

In 29 patients all videos were available for analysis. Vascular abnormalities were scored most frequently in HD + i-scan2 bronchoscopy (1.33 ± 0.29 abnormal or suspicious sites per patient) as compared to 0.12 ± 0.05 site for AFB (P = 0.003). Sites suspicious for preinvasive lesions were most frequently reported using AFB (0.74 ± 0.12 sites per patient) as compared to 0.17 ± 0.06 for both WLB and HD bronchoscopy (P = 0.003). Tumors were detected equally by all modalities. The preferred modality was HD bronchoscopy with i-scan (tone- plus surface and surface enhancement in respectively 38% and 35% of cases P = 0.006).

Conclusions

This study shows that high definition bronchoscopy with image enhancement technique may result in better detection of subtle vascular abnormalities in the airways. Since these abnormalities may be related to preneoplastic lesions and tumors this is of clinical relevance. Further investigations using this technique relating imaging to histology are warranted.     

Mutations of human cytochrome P450 reductase differentially modulate heme oxygenase-1 activity and oligomerization

Genetic variations in POR, encoding NADPH-cytochrome P450 oxidoreductase (CYPOR), can diminish the function of numerous cytochromes P450, and also have the potential to block degradation of heme by heme oxygenase-1 (HO-1). Purified full-length human CYPOR, HO-1, and biliverdin reductase were reconstituted in lipid vesicles and assayed for NADPH-dependent conversion of heme to bilirubin. Naturally-occurring human CYPOR variants queried were: WT, A115V, Y181D, P228L, M263V, A287P, R457H, Y459H, and V492E. All CYPOR variants exhibited decreased bilirubin production relative to WT, with a lower apparent affinity of the CYPOR–HO-1 complex than WT. Addition of FMN or FAD partially restored the activities of Y181D, Y459H, and V492E. When mixed with WT CYPOR, only the Y181D CYPOR variant inhibited heme degradation by sequestering HO-1, whereas Y459H and V492E were unable to inhibit HO-1 activity suggesting that CYPOR variants might have differential binding affinities with redox partners. Titrating the CYPOR–HO-1 complex revealed that the optimal CYPOR:HO-1 ratio for activity was 1:2, lending evidence in support of productive HO-1 oligomerization, with higher ratios of CYPOR:HO-1 showing decreased activity. In conclusion, human POR mutations, shown to impact P450 activities, also result in varying degrees of diminished HO-1 activity, which may further complicate CYPOR deficiency.     

Mismatch Responses in the Awake Rat: Evidence from Epidural Recordings of Auditory Cortical Fields

Detecting sudden environmental changes is crucial for the survival of humans and animals. In the human auditory system the mismatch negativity (MMN), a component of auditory evoked potentials (AEPs), reflects the violation of predictable stimulus regularities, established by the previous auditory sequence. Given the considerable potentiality of the MMN for clinical applications, establishing valid animal models that allow for detailed investigation of its neurophysiological mechanisms is important. Rodent studies, so far almost exclusively under anesthesia, have not provided decisive evidence whether an MMN analogue exists in rats. This may be due to several factors, including the effect of anesthesia. We therefore used epidural recordings in awake black hooded rats, from two auditory cortical areas in both hemispheres, and with bandpass filtered noise stimuli that were optimized in frequency and duration for eliciting MMN in rats. Using a classical oddball paradigm with frequency deviants, we detected mismatch responses at all four electrodes in primary and secondary auditory cortex, with morphological and functional properties similar to those known in humans, i.e., large amplitude biphasic differences that increased in amplitude with decreasing deviant probability. These mismatch responses significantly diminished in a control condition that removed the predictive context while controlling for presentation rate of the deviants. While our present study does not allow for disambiguating precisely the relative contribution of adaptation and prediction error processing to the observed mismatch responses, it demonstrates that MMN-like potentials can be obtained in awake and unrestrained rats.     

Suitability of Pharmacokinetic Models for Dynamic Contrast-Enhanced MRI of Abdominal Aortic Aneurysm Vessel Wall: A Comparison

Purpose

Increased microvascularization of the abdominal aortic aneurysm (AAA) vessel wall has been related to AAA progression and rupture. The aim of this study was to compare the suitability of three pharmacokinetic models to describe AAA vessel wall enhancement using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI).

Materials and Methods

Patients with AAA underwent DCE-MRI at 1.5 Tesla. The volume transfer constant (K^trans), which reflects microvascular flow, permeability and surface area, was calculated by fitting the blood and aneurysm vessel wall gadolinium concentration curves. The relative fit errors, parameter uncertainties and parameter reproducibilities for the Patlak, Tofts and Extended Tofts model were compared to find the most suitable model. Scan-rescan reproducibility was assessed using the interclass correlation coefficient and coefficient of variation (CV). Further, the relationship between K^trans and AAA size was investigated.

Results

DCE-MRI examinations from thirty-nine patients (mean age±SD: 72±6 years; M/F: 35/4) with an mean AAA maximal diameter of 49±6 mm could be included for pharmacokinetic analysis. Relative fit uncertainties for K^trans based on the Patlak model (17%) were significantly lower compared to the Tofts (37%) and Extended Tofts model (42%) (p<0.001). K^trans scan-rescan reproducibility for the Patlak model (ICC = 0.61 and CV = 22%) was comparable with the Tofts (ICC = 0.61, CV = 23%) and Extended Tofts model (ICC = 0.76, CV = 22%). K^trans was positively correlated with maximal AAA diameter (Spearman’s ρ = 0.38, p = 0.02) using the Patlak model.

Conclusion

Using the presented imaging protocol, the Patlak model is most suited to describe DCE-MRI data of the AAA vessel wall with good K^trans scan-rescan reproducibility.     

Parallel strategies for the preparation and selection of liver-targeted glucocorticoid receptor antagonists

Libraries of mifepristone analogs, MP-Acids, were designed and synthesized to increase the chances of identifying GR antagonists that possess liver-selective pharmacological profiles. MP-Acids were uniformly potent GR antagonists in binding and in cell-based functional assays. A high throughput pharmacokinetic selection strategy that employs the cassette dosing of MP-Acids was developed to identify liver-targeting compounds. Thus, resource-intensive in vivo assays to measure liver-selective pharmacology were enriched with GR antagonists that achieve high concentrations in the liver.     

Pyrrolidine-constrained phenethylamines: The design of potent, selective, and pharmacologically efficacious dipeptidyl peptidase IV (DPP4) inhibitors from a lead-like screening hit

A novel series of pyrrolidine-constrained phenethylamines were developed as dipeptidyl peptidase IV (DPP4) inhibitors for the treatment of type 2 diabetes. The cyclohexene ring of lead-like screening hit 5 was replaced with a pyrrolidine to enable parallel chemistry, and protein co-crystal structural data guided the optimization of N-substituents. Employing this strategy, a >400x improvement in potency over the initial hit was realized in rapid fashion. Optimized compounds are potent and selective inhibitors with excellent pharmacokinetic profiles. Compound 30 was efficacious in vivo, lowering blood glucose in ZDF rats that were allowed to feed freely on a mixed meal.