Phloem Mobility of Xenobiotics: II. Bioassay Testing of the Unified Mathematical Model

Two bioassays were used to test phloem mobility of selected xenobiotic compounds: (a) excised bean leaf assay; (b) rooted bean leaf assay. Compounds assayed were N-alkylpyridiniums with systematic variation in octanol-water partition coefficients (log K_ow), substituted benzoic acids with about the same log K_ow value but variable acidities. Results of the assays strongly conform, quantitatively, to the predictions of the unified mathematical model. Results also indicate that the membrane permeability value of a compound, which depends directly on log K_ow value, is the overriding factor in determining phloem mobility. When the weak acid functionality of a compound confers increased phloem mobility, it does so principally by making the log K_ow value, and consequently the membrane permeability of the compound more optimal.     

Distribution of opioidergic,sympathetic and neuropeptide Y-positive nerves in the sphincter of Oddi and biliary tree of the monkey,Macaca fascicularis

Summary The opioidergic, sympathetic and neuropeptide Y-positive innervation of the sphincter of Oddi (common bile duct sphincter and pancreatic duct sphincter), as well as other segments of the extrahepatic biliary tree was studied in the monkey by use of immunohistochemistry. Methionine-enkephalin-positive nerves were seen to innervate the smooth muscle of all portions of the sphincter of Oddi and also local ganglion cells. No methionine-enkephalin-positive nerves could be detected in the common bile duct, pancreatic duct or gallbladder. Tyrosine hydroxylase-positive nerves occurred between smooth muscle bundles and also ran to local ganglion cells as well as along the common bile duct. Neuropeptide Y-positive nerves were observed within smooth muscle of the sphincter of Oddi (all portions), common bile duct, pancreatic duct and gallbladder. No evidence of any differential innervation of the pancreatic duct and common bile duct sphincters could be detected with these markers.     

Ribosome-bound eukaryotic elongation factor 2 protects 5 S rRNA from modification.

The interaction between eukaryotic elongation factor eEF-2 and reconstituted 80 S ribosomes was investigated by analyzing the accessibility of 5 S ribosomal RNA for chemical and enzymatic modification. Ribosomes reconstituted from derived subunits were modified, and the positions of the modified sites were identified by primer extension using a 5 S rRNA-specific probe. All reactive sites were located between nucleotides 38 and 99, and most of them were found in putative single-stranded regions of the 5 S rRNA. Conversion of the ribosomes to the post-translocation type of particles by treatment with the translational inhibitor ricin resulted in the exposure of 3 additional bases for chemical modification, suggesting that the 5 S rRNA was more exposed in this type of ribosome. After binding of eEF-2 in complex with the non-hydrolyzable GTP analogue guanosine 5'-(beta, gamma-methylene)-triphosphate, most of the exposed bases in the 5 S rRNA were protected against both chemical and enzymatic modification.     

Tryptamine derivatives disarm colistin resistance in polymyxin-resistant gram-negative bacteria

The last three decades have seen a dwindling number of novel antibiotic classes approved for clinical use and a concurrent increase in levels of antibiotic resistance, necessitating alternative methods to combat the rise of multi-drug resistant bacteria. A promising strategy employs antibiotic adjuvants, non-toxic molecules that disarm antibiotic resistance. When co-dosed with antibiotics, these compounds restore antibiotic efficacy in drug-resistant strains. Herein we identify derivatives of tryptamine, a ubiquitous biochemical scaffold containing an indole ring system, capable of disarming colistin resistance in the Gram-negative bacterial pathogens Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli while having no inherent bacterial toxicity. Resistance was overcome in strains carrying endogenous chromosomally-encoded colistin resistance machinery, as well as resistance conferred by the mobile colistin resistance-1 (mcr-1) plasmid-borne gene. These compounds restore a colistin minimum inhibitory concentration (MIC) below the Clinical & Laboratory Sciences Institute (CLSI) breakpoint in all resistant strains.     

Pea Lectin Expressed Transgenically in Oilseed Rape Reduces Growth Rate of Pollen Beetle Larvae

In several studies plant lectins have shown promise as transgenic resistance factors against various insect pests. We have here shown that pea seed lectin is a potential candidate for use against pollen beetle, a serious pest of Brassica oilseeds. In feeding assays where pollen beetle larvae were fed oilseed rape anthers soaked in a 1% solution of pea lectin there was a reduction in survival of 84% compared to larvae on control treatment and the weight of surviving larvae was reduced by 79%. When a 10% solution of pea lectin was used all larvae were dead after 4 days of testing. To further evaluate the potential use of pea lectin, transgenic plants of oilseed rape (Brassica napus cv. Westar) were produced in which the pea lectin gene under control of the pollen-specific promoter Sta44-4 was introduced. In 11 out of 20 tested plants of the T₀-generation there was a significant reduction in larval weight, which ranged up to 46% compared to the control. A small but significant reduction in larval survival rate was also observed. In the T₂-generation significant weight reductions, with a maximum of 32%, were obtained in 10 out of 33 comparisons between transgenic plants and their controls. Pea lectin concentrations in anthers of transgenic T₂-plants ranged up to 1.5% of total soluble protein. There was a negative correlation between lectin concentration and larval growth. Plants from test groups with significant differences in larval weights had a significantly higher mean pea lectin concentration, 0.64% compared to 0.15% for plants from test groups without effect on larval weight. These results support the conclusion that pea lectin is a promising resistance factor for use in Brassica oilseeds against pollen beetles.     

Down-regulation of Rac activity during beta 2 integrin-mediated adhesion of human neutrophils

In human neutrophils, beta2 integrin engagement mediated a decrease in GTP-bound Rac1 and Rac2. Pretreatment of neutrophils with LY294002 or PP1 (inhibiting phosphatidylinositol 3-kinase (PI 3-kinase) and Src kinases, respectively) partly reversed the beta2 integrin-induced down-regulation of Rac activities. In contrast, beta2 integrins induced stimulation of Cdc42 that was independent of Src family members. The PI 3-kinase dependence of the beta2 integrin-mediated decrease in GTP-bound Rac could be explained by an enhanced Rac-GAP activity, since this activity was blocked by LY204002, whereas PP1 only had a minor effect. The fact that only Rac1 but not Rac2 (the dominating Rac) redistributed to the detergent-insoluble fraction and that it was independent of GTP loading excludes the possibility that down-regulation of Rac activities was due to depletion of GTP-bound Rac from the detergent-soluble fraction. The beta2 integrin-triggered relocalization of Rac1 to the cytoskeleton was enabled by a PI 3-kinase-induced dissociation of Rac1 from LyGDI. The dissociations of Rac1 and Rac2 from LyGDI also explained the PI 3-kinase-dependent translocations of Rac GTPases to the plasma membrane. However, these accumulations of Rac in the membrane, as well as that of p47phox and p67phox, were also regulated by Src tyrosine kinases. Inasmuch as Rac GTPases are part of the NADPH oxidase and the respiratory burst is elicited in neutrophils adherent by beta2 integrins, our results indicate that activation of the NADPH oxidase does not depend on the levels of Rac-GTP but instead requires a beta2 integrin-induced targeting of the Rac GTPases as well as p47phox and p67phox to the plasma membrane.     

Cystatin C Is Not Causally Related to Coronary Artery Disease

Background

Strong and independent associations between plasma concentration of cystatin C and risk of cardiovascular disease (CVD) suggests causal involvement of cystatin C.

Aim

The aim of our study was to assess whether there is a causal relationship between plasma concentration of cystatin C and risk of coronary artery disease (CAD) using a Mendelian Randomization approach.

Methods

We estimated the strength of association of plasma cystatin C on CAD risk and the strength of association of the strongest GWAS derived cystatin C SNP (rs13038305) on plasma cystatin C in the population-based Malmö Diet and Cancer Study (MDC) and thereafter the association between rs13038305 and CAD in the MDC (3200 cases of CAD and 24418 controls) and CARDIOGRAM (22233 cases of CAD and 64762 controls).

Results

Each standard deviation (SD) increment of plasma cystatin C was associated with increased risk of CAD (OR = 1.20, 95% CI 1.07–1.34) after full adjustment. Each copy of the major allele of rs13038305 was associated with 0.34 SD higher plasma concentration of cystatin C (P<1 x 10^-35), resulting in a power of >98% to detect a significant relationship between rs13038305 and CAD in MDC and CARDIOGRAM pooled. The odds ratio for CAD (per copy of the major rs13038305 allele) was 1.00 (0.94–1.07); P = 0.92 in MDC, 0.99 (0.96–1.03); P = 0.84 in CARDIOGRAM and 1.00 (0.97–1.03); P = 0.83 in MDC and CARDIOGRAM pooled.

Conclusion

Genetic elevation of plasma cystatin C is not related to altered risk of CAD, suggesting that there is no causal relationship between plasma cystatin C and CAD. Rather, the association between cystatin C and CAD appears to be due to the association of eGFR and CAD.     

The Early-Onset Myocardial Infarction Associated PHACTR1 Gene Regulates Skeletal and Cardiac Alpha-Actin Gene Expression

The phosphatase and actin regulator 1 (PHACTR1) locus is a very commonly identified hit in genome-wide association studies investigating coronary artery disease and myocardial infarction (MI). However, the function of PHACTR1 in the heart is still unknown. We characterized the mechanisms regulating Phactr1 expression in the heart, used adenoviral gene delivery to investigate the effects of Phactr1 on cardiac function, and analyzed the relationship between MI associated PHACTR1 allele and cardiac function in human subjects. Phactr1 mRNA and protein levels were markedly reduced (60%, P<0.01 and 90%, P<0.001, respectively) at 1 day after MI in rats. When the direct myocardial effects of Phactr1 were studied, the skeletal α-actin to cardiac α-actin isoform ratio was significantly higher (1.5-fold, P<0.05) at 3 days but 40% lower (P<0.05) at 2 weeks after adenovirus-mediated Phactr1 gene delivery into the anterior wall of the left ventricle. Similarly, the skeletal α-actin to cardiac α-actin ratio was lower at 2 weeks in infarcted hearts overexpressing Phactr1. In cultured neonatal cardiac myocytes, adenovirus-mediated Phactr1 overexpression for 48 hours markedly increased the skeletal α-actin to cardiac α-actin ratio, this being associated with an enhanced DNA binding activity of serum response factor. Phactr1 overexpression exerted no major effects on the expression of other cardiac genes or LV structure and function in normal and infarcted hearts during 2 weeks’ follow-up period. In human subjects, MI associated PHACTR1 allele was not associated significantly with cardiac function (n = 1550). Phactr1 seems to regulate the skeletal to cardiac α-actin isoform ratio.     

Modulating the development of E. coli biofilms with 2-aminoimidazoles

The synthesis of a 20 member 2-aminoimidazole/triazole pilot library is reported. Each member of the library was screened for its ability to inhibit or promote biofilm development of either Escherichia coli and Acinetobacter baumannii. From this screen, E. coli-selective 2-aminoimidazoles were discovered, with the best inhibitor inhibiting biofilm development with an IC₅₀ of 13 μM. The most potent promoter of E. coli biofilm formation promoted biofilm development by 321% at 400 μM.     

EVITA: a tool for the early EValuation of pharmaceutical Innovations with regard to Therapeutic Advantage

Background

New drugs are generally claimed to represent a therapeutic innovation. However, scientific evidence of a substantial clinical advantage is often lacking. This may be the result of using inadequate control groups or surrogate outcomes only in the clinical trials. In view of this, EVITA was developed as a user-friendly transparent tool for the early evaluation of the additional therapeutic value of a new drug.

Methods

EVITA does not evaluate a new compound per se but in an approved indication in comparison with existing therapeutic strategies. Placebo as a comparator is accepted only in the absence of an established therapy or if employed in an add-on strategy on top. The evaluation attributes rating points to the drug in question, taking into consideration both therapeutic benefit and risk profile. The compound scores positive points for superiority in efficiency and/or adverse effects as demonstrated in randomized controlled trials (RCTs), whilst negative points are awarded for inferiority and/or an unfavorable risk profile. The evaluation follows an algorithm considering the clinical relevance of the outcomes, the strength of the therapeutic effect and the number of RCTs performed. Categories for therapeutic aim and disease severity, although essential parts of the EVITA assessment, are attributed but do not influence the EVITA score which is presented as a color-coded bar graph. In case the available data were unsuitable for an EVITA calculation, a traffic-type yield sign is assigned instead to criticize such practice. The results are presented online http://www.evita-report.de together with all RCTs considered as well as the reasons for excluding a given RCT from the evaluation. This allows for immediate revision in response to justified criticism and simplifies the inclusion of new data.

Results

As examples, four compounds which received approval within the last years were evaluated for one of their clinical indications: lenalidomide, pioglitazone, bupropion and zoledronic acid. Only the first achieved an EVITA score above zero indicating therapeutic advantage.

Conclusions

The strength of EVITA appears to lie in its speedy assessment of the potential therapeutic advantage of a new drug for a given indication. At the same time, this approach draws attention to the typical deficits of data used for drug approval. EVITA is not intended to replace classical health technology assessment reports but rather serves as a screening tool in the sense of horizon scanning.