Metabolism of retinoids and arachidonic acid by human and mouse cytochrome P450 1b1.

The cytochrome P450 family 1 (CYP1) is considered to be one of the xenobiotic-metabolizing enzyme families and is responsible for oxidative metabolism of polycyclic aromatic hydrocarbons. For example, mouse Cyp1b1 was originally identified as the enzyme responsible for oxidative metabolism of 7,12-dimethylbenz(alpha)anthracene (DMBA). A comparison of the kinetics of this metabolism by mouse and human CYP1B1 orthologs revealed the mouse enzyme to have a more favorable metabolism of DMBA, with a catalytic efficiency ratio (CER) of 0.23. However, CYP1 enzymes are also capable of metabolism of endobiotics, and in the present study, the metabolism of retinoids and lipid endobiotics by human CYP1B1 and mouse Cyp1b1 orthologs was compared. Both hemoproteins oxidized retinol to retinal and retinal to retinoate, but did not oxidize retinoate. The CYP1B1 to Cyp1b1 CERs were 13 and 26 for the two steps, respectively; the Cyp1b1 K(m(app)) values for retinoids were 20-fold higher. Human family 1 cytochromes P450 had unique regional specificities for arachidonate oxidation: the major metabolites of CYP1A1, CYP1A2, and CYP1B1 were 75% terminal hydroxyeicosatetraenoic fatty acids (HETEs), 52% epoxyeicosatrienoic fatty acids (EETs), and 54% mid-chain HETEs, respectively. CYP1A1 and CYP1B1 K(m(app)) values for arachidonate were about 30 microM, whereas CYP1A2 K(m(app)) was 95 microM. The major metabolites of arachidonic acid by Cyp1b1 were EETs (50%) and midchain HETEs (37%). The mouse ortholog had a CER for metabolite production of 64 due to a K(m(app)) of 0.5 mM for arachidonate.     

Base-promoted direct amidation of esters: beyond the current scope and practical applications

The base-promoted direct amidation of unactivated esters is among the most useful reactions for amide bond formation in contemporary organic chemistry. The intensive research in this area has led to the development of a number of new methods to achive this transformation. However, to date, the existing literature is more methodological and in many instances lacks practical directions. Therefore, the full potential of this transformation is yet to be revealed by broadening the substrate scope. In a search for new practical applications of the amidation reaction, herein we present a comprehensive study of a number of base-promoted direct amidations that encompass a wide range of amines and esters. Furthermore, we applied our findings in the synthesis of phosphoramidates and several industrially relevant products.

A new method for direct amidation of unactivated esters with unprecedented substrate scope is developed.     

Non-linear renal function decline is frequent in patients with type 2 diabetes who progress fast to end-stage renal disease and is associated with African-Caribbean ethnicity and HbA1c variability

To our knowledge, there are no studies examining eGFR trajectories in an ethnically diverse cohort of T2DM patients with established DKD and long follow-up. We conducted a retrospective analysis of medical records of T2DM patients attending a specialist diabetes renal clinic in order to identify risk factors and specific eGFR trajectories associated with ESRD. There is limited information and long term follow-up on eGFR trajectories in ethnically diverse cohorts of T2DM patients with established diabetic kidney disease. We conducted a retrospective analysis of medical records of 398 T2DM patients (46.5% African-Carribean ethnicity) to identify risk factors and specific eGFR trajectories associated with end-stage renal disease (ESRD). A non-linear eGFR trajectory was observed in 59% of the 71 patients who reached ESRD. African-Caribbean ethnicity and glycaemic variability are independently associated with distinct non-linear eGFR trajectories that result in fast progression to ESRD. Clinicians should be aware that non-linear eGFR decline is frequent in patients with T2DM who have fast progression to ESRD. Predicting renal function decline based on patterns and early changes in eGFR trajectories and associated risk factors, may better enable individualized risk stratification and care for those at highest risk of rapid progression to ESRD.     

Deletion of the gene encoding p60 in Listeria monocytogenes leads to abnormal cell division and loss of actin-based motility

Protein p60 encoded by the iap gene is regarded as an essential gene product of Listeria monocytogenes. Here we report, however, the successful construction of a viable iap deletion mutant of L. monocytogenes EGD. The mutant, which produces no p60, shows abnormal septum formation and tends to form short filaments and hooked forms during logarithmic growth. These abnormal bacterial cells break into almost normal sized single bacteria in the late-stationary-growth phase. The iap mutant is strongly attenuated in a mouse model after intravenous injection, demonstrating the importance of p60 during infection, and the invasiveness of the Deltaiap mutant for 3T6 fibroblasts and Caco-2 epithelial cells is slightly reduced. Upon uptake by epithelial cells and macrophages, the iap mutant escapes from the phagosome into the cytosol with the same efficiency as the wild-type strain, and the mutant bacteria also grow intracellularly at a rate similar to that of the wild-type strain. Intracellular movement and cell-to-cell spread are drastically reduced in various cell lines, since the iap-negative bacteria fail to induce the formation of actin tails. However, the bacteria are covered with actin filaments. Most intracellular bacteria show a nonpolar and uneven distribution of ActA around the cell, in contrast to that for the wild-type strain, where ActA is concentrated at the old pole. In an iap(+) revertant strain that produces wild-type levels of p60, intracellular movement, cell-to-cell spread, and polar distribution of ActA are fully restored. In vitro analysis of ActA distribution on the filaments of the Deltaiap strain shows that the loss of bacterial septum formation leads to ActA accumulation at the presumed division sites. In the light of data presented here and elswhere, we propose to rename iap (invasion-associated protein) cwhA (cell wall hydrolase A).     

The process of incorporating insulin pumps into the everyday lives of people with Type 1 diabetes: A critical interpretive synthesis

Background

Insulin pump therapy (IPT) is a technological advancement that has been developed to help people manage Type 1 diabetes (T1D). However, ways of managing diabetes requiring the implementation of health technologies bring new complexities and a need to understand the factors which enable people with T1D to incorporate a novel device. This new comprehension could provide an exemplar for people with long‐term conditions to incorporate new technologies more generally.

Objective

To determine what influences the incorporation, adaptation and use of IPT into the everyday lives of people living with diabetes.

Design

Critical interpretive synthesis (CIS) using systematic searches undertaken in 7 electronic databases of literature, published 2008 onwards.

Results

A total of 4998 titles were identified, 274 abstracts reviewed, 39 full articles retrieved and 22 papers selected for analysis. Three themes emerged which were of relevance to the introduction and use of IPT; Tensions between expectations and experiences in adoption and early adaptation; Negotiation of responsibility and accessing support; Reflexivity, active experimentation and feedback.

Conclusions

This CIS builds on earlier reviews on lived experiences of IPT. Novel insights are offered through examination of the experiences of pump users from children through to adults, their families and health‐care professionals. Expectations of what the device can do to improve self‐management impacts on the early stages of adoption as the reality of the technology requires substantial thought and action. Areas for intervention to improve IPT incorporation include establishing who is responsible for management tasks of the device and enabling navigation to further means of support and resources.     

Molecular genetics of primary congenital glaucoma in Brazil

PURPOSE: To determine the distribution of CYP1B1 gene mutations in Brazilian patients with primary congenital glaucoma (PCG). METHODS: PCG diagnosis was established by presence of buphthalmos in at least one affected eye and associated high intraocular pressures before the age of 3 years. CYP1B1 mutation screening of 52 patients with PCG was performed by SSCP and direct sequencing of PCR fragments. RESULTS: Eleven mutations, four of which are novel, were observed in 26 (50%) individuals. A new frameshift mutation (4340delG) was observed in 20.2% of all individuals screened. These individuals had early-onset, bilateral glaucoma that necessitated multiple surgical interventions. CYP1B1 mutations were twice as frequent in affected individuals of European descent as in individuals of African descent. Analysis of six intragenic single nucleotide polymorphisms (SNPs) established 5'-C-C-G-G-T-A-3' as the most common haplotype among the affected Brazilian individuals. A nonsense mutation (W57X) previously reported in an individual with Peters anomaly (compound heterozygote) was also observed in two individuals with PCG but combined with different mutations. A newly developed SSCP assay enabled us to detect all DNA mutations and polymorphisms previously detected by direct sequencing. CONCLUSIONS: Our results indicate that CYP1B1 mutations may be responsible for half of cases of PCG in the Brazilian population. The SNP haplotype 5'-C-C-G-G-T-A-3' was associated with the majority of CYP1B1 mutations. This haplotype harbors the high-activity V432 allele, which is emerging as a putative susceptibility factor in several cancers.