alpha,alpha'-disubstituted amino acids with silylated side chains as lipophilic building blocks for the synthesis of peptaibol analogues

New alpha,alpha'-disubstituted amino acids with silylated side chains have been synthesized in racemic form. Starting from a Schiff base of glycine tert-butyl ester, a large variety of alpha,alpha'-dialkylated amino acids has been obtained, depending on the alkylating reagents. The application of a hydrosilylation methodology enabled the synthesis of the same unnatural amino acids in an enantiomerically pure form. The ability of these bulky amino acids to be incorporated into peptides by solution-phase methodology has also been demonstrated, since constrained silylated dipeptides have been synthesized. These new lipophilic building blocks could be useful and innovative in the design of peptaibol analogues.     

The interplay of RecA-related proteins and the MND1-HOP2 complex during meiosis in Arabidopsis thaliana

During meiosis, homologous chromosomes recognize each other, align, and exchange genetic information. This process requires the action of RecA-related proteins Rad51 and Dmc1 to catalyze DNA strand exchanges. The Mnd1-Hop2 complex has been shown to assist in Dmc1-dependent processes. Furthermore, higher eukaryotes possess additional RecA-related proteins, like XRCC3, which are involved in meiotic recombination. However, little is known about the functional interplay between these proteins during meiosis. We investigated the functional relationship between AtMND1, AtDMC1, AtRAD51, and AtXRCC3 during meiosis in Arabidopsis thaliana. We demonstrate the localization of AtMND1 to meiotic chromosomes, even in the absence of recombination, and show that AtMND1 loading depends exclusively on AHP2, the Arabidopsis Hop2 homolog. We provide evidence of genetic interaction between AtMND1, AtDMC1, AtRAD51, and AtXRCC3. In vitro assays suggest that this functional link is due to direct interaction of the AtMND1-AHP2 complex with AtRAD51 and AtDMC1. We show that AtDMC1 foci accumulate in the Atmnd1 mutant, but are reduced in number in Atrad51 and Atxrcc3 mutants. This study provides the first insights into the functional differences of AtRAD51 and AtXRCC3 during meiosis, demonstrating that AtXRCC3 is dispensable for AtDMC1 focus formation in an Atmnd1 mutant background, whereas AtRAD51 is not. These results clarify the functional interactions between key players in the strand exchange processes during meiotic recombination. Furthermore, they highlight a direct interaction between MND1 and RAD51 and show a functional divergence between RAD51 and XRCC3.     

Study of a lipophilic captopril analogue binding to angiotensin I converting enzyme

Human ACE is a central component of the renin–angiotensin system and a major therapeutic target for cardiovascular diseases. The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a zinc active site with similar but distinct substrate and inhibitor specificities. In this study, we present the biological activity of silacaptopril, a silylated analogue of captopril, and its binding affinity towards ACE. Based on the recently determined crystal structures of both the ACE domains, a series of docking calculations were carried out in order to study the structural characteristics and the binding properties of silacaptopril and its analogues with ACE. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Detection By Whole-Genome Sequencing of a Novel Metallo-β-Lactamase Produced By Wautersiella Falsenii Causing Urinary Tract Infection in Tunisia

Wautersiella falsenii is a rarely non-fermenting Gram-negative bacterium and belongs to the Flavobacteriaceae family. This nosocomial pathogen can cause several human infections, especially among immunocompromised patients. Here, we describe the whole genome sequence of a clinical W. falsenii strain isolated from a urine sample of a 35-year-old woman with a urinary tract infection in Tunisia. We investigated its phenotype and genotype.After bacterial identification by the MALDI-TOF method, the whole-genome sequencing of this strain was performed. This isolate was not susceptible to various antibiotics, including β-lactams, aminoglycosides, and quinolones. However, it remains susceptible to imipenem (MIC = 0.25 mg/l), ertapenem (MIC = 0.75 mg/l), and meropenem (MIC = 0.19 mg/l). Interestingly, the E-TEST^® (MP/MPI) showed a reduced MIC of meropenem +/− EDTA (0.064 μg/ml). Besides, the color change from yellow to red in the β CARBA test only after 24 hours of incubation can be interpreted in two ways. On the one hand, as a likely low expression of the gene encoding metallo-β-lactamase. On the other hand, and more likely, it may be a false-positive result because, according to the test manufacturer''s recommendations, the test should be read after 30 minutes. Perhaps, therefore, this gene is not expressed in the tested strain. Moreover, the whole-genome sequence analysis demonstrated the presence of a novel chromosomally located subclass B1 metallo-β-lactamase EBR-like enzyme, sharing 94.92% amino acid identity with a previously described carbapenemase produced by Empedobacter brevis, EBR-1. The results also showed the detection of other antibiotic resistance genes and the absence of plasmids. So far, this study is the first report on the detection of W. falsenii in Tunisia. These findings prove that W. falsenii could be a potential reservoir of antibiotic resistance genes, e.g., β-lactamases. Collaborative efforts and effective hygiene measures should be established to prevent the emergence of this species in our health care settings.     

Diabetic kidney disease in FVB/NJ Akita mice: temporal pattern of kidney injury and urinary nephrin excretion

Akita mice are a genetic model of type 1 diabetes. In the present studies, we investigated the phenotype of Akita mice on the FVB/NJ background and examined urinary nephrin excretion as a marker of kidney injury. Male Akita mice were compared with non-diabetic controls for functional and structural characteristics of renal and cardiac disease. Podocyte number and apoptosis as well as urinary nephrin excretion were determined in both groups. Male FVB/NJ Akita mice developed sustained hyperglycemia and albuminuria by 4 and 8 weeks of age, respectively. These abnormalities were accompanied by a significant increase in systolic blood pressure in 10-week old Akita mice, which was associated with functional, structural and molecular characteristics of cardiac hypertrophy. By 20 weeks of age, Akita mice developed a 10-fold increase in albuminuria, renal and glomerular hypertrophy and a decrease in the number of podocytes. Mild-to-moderate glomerular mesangial expansion was observed in Akita mice at 30 weeks of age. In 4-week old Akita mice, the onset of hyperglycemia was accompanied by increased podocyte apoptosis and enhanced excretion of nephrin in urine before the development of albuminuria. Urinary nephrin excretion was also significantly increased in albuminuric Akita mice at 16 and 20 weeks of age and correlated with the albumin excretion rate. These data suggest that: 1. FVB/NJ Akita mice have phenotypic characteristics that may be useful for studying the mechanisms of kidney and cardiac injury in diabetes, and 2. Enhanced urinary nephrin excretion is associated with kidney injury in FVB/NJ Akita mice and is detectable early in the disease process.     

Vitamin D Deficiency Reduces Vascular Reactivity of Coronary Arterioles in Male Rats

Background: Vitamin D deficiency (VDD) may be considered an independent cardiovascular (CV) risk factor, and it is well known that CV risk is higher in males. Our goal was to investigate the pharmacological reactivity and receptor expression of intramural coronary artery segments of male rats in cases of different vitamin D supply. Methods: Four-week-old male Wistar rats were divided into a control group (n = 11) with optimal vitamin D supply (300 IU/kgbw/day) and a VDD group (n = 11, <0.5 IU/kgbw/day). After 8 weeks of treatment, intramural coronary artery segments were microprepared, their pharmacological reactivity was examined by in vitro microangiometry, and their receptor expression was investigated by immunohistochemistry. Results: Thromboxane A₂ (TXA₂)-agonist induced reduced vasoconstriction, testosterone (T) and 17-β-estradiol (E2) relaxations were significantly decreased, a significant decrease in thromboxane receptor (TP) expression was shown, and the reduction in estrogen receptor-α (ERα) expression was on the border of significance in the VDD group. Conclusions: VD-deficient male coronary arteries showed deteriorated pharmacological reactivity to TXA₂ and sexual steroids (E2, T). Insufficient vasoconstrictor capacity was accompanied by decreased TP receptor expression, and vasodilator impairments were mainly functional. The decrease in vasoconstrictor and vasodilator responses results in narrowed adaptational range of coronaries, causing inadequate coronary perfusion that might contribute to the increased CV risk in VDD.     

Modulation of 1alpha,25-dihydroxyvitamin D3-membrane associated, rapid response steroid binding protein expression in mouse odontoblasts by 1alpha,25-(OH)2D3

The rapid, nongenomic effects of 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3 have been related to a 1,25D3-membrane associated, rapid response steroid binding protein or 1,25D3-[MARRS]bp, with a molecular weight of 65 kDa, in several tissues and species. Currently, no information is available concerning the nongenomic responses to 1alpha,25-(OH)2D3 in dental tissues. In order to investigate the expression of 1,25D3-[MARRS]bp in dental cells, in the presence or absence of 1alpha,25-(OH)2D3, we have used rabbit polyclonal antibodies directed against the N-terminus of the 1,25D3-[MARRS]bp (Ab099) that recognizes the 1alpha,25-(OH)2D3 binding protein in chick intestinal basolateral membranes and a mouse odontoblast-like cell line (MO6-G3). Western blotting and flow cytometric analyses with Ab099 specifically detected 1,25D3-[MARRS]bp in MO6-G3 cells. Moreover, 1,25D3-[MARRS]bp was up-regulated, in vivo, in differentiated dental cells. Electron microscopic analysis confirmed the plasma membrane localization of this binding protein and also showed its intracellular presence. Incubation of MO6-G3 cells with different doses of 1alpha,25-(OH)2D3 for 36 h resulted in an inhibition of 1,25D3-[MARRS]bp expression with a maximal effect at 50 nM steroid. In addition, the culture media of MO6-G3 cells contains immunoreactive 1,25D3-[MARRS]bp. Immunogold positive membrane vesicle-like structures are present in the extracellular matrix of MO6-G3 cells. Altogether, these results indicate that the 1,25D3-[MARRS]bp expression in MO6-G3 cells is modulated by 1alpha,25-(OH)2D3. In conclusion, this 1alpha,25-(OH)2D3 binding protein could play an important role in the rapid, nongenomic responses to 1alpha,25-(OH)2D3 in dental cells.     

Metabolism in orange fruits is driven by photooxidative stress in the leaves

In plants, stress signals propagate to trigger distant responses and thus stress acclimation in non‐exposed organs. We tested here the hypothesis that leaves submitted to photooxidative stress may influence the metabolism of nearby fruits and thus quality criteria. Leaves of orange trees (Citrus sinensis (L.) Osbeck cv. ‘Navelate’) were acclimated to shade for 1 week and then submitted to full (FL) and medium light (ML) conditions. As expected, photoinhibition was detected in leaves of both FL and ML treatments as revealed by stress indicators (F_v/F_m, Performance Index) for at least 99 h after treatments. In the fruits near the stressed leaves, we then determined the activities of enzymes related to oxidative stress, superoxide dismutase, catalase and the enzymes of the ascorbate (AA)/glutathione cycle, as well as the contents in sugars, organic acids and carotenoids. Ascorbate peroxidase and monodehydroascorbate reductase activities in the pulp of fruits were dramatically higher in both treatments when compared to the control. AA and total sugars were not affected by the photooxidative stress. However, the FL treatment resulted in a 16% increase in total organic acids, with succinic acid being the major contributor, a shift towards less glucose + fructose and more sucrose, and a 15% increase in total carotenoids, with cis‐violaxanthin being the major contributor. Our observations strongly suggest the existence of a signal generated in leaves in consequence of photooxidative stress, transmitted to nearby fruits. Exploiting such a signal by agronomic means promises exciting perspectives in managing quality criteria in fruits accumulating carotenoids.