Arhgef15 Promotes Retinal Angiogenesis by Mediating VEGF-Induced Cdc42 Activation and Potentiating RhoJ Inactivation in Endothelial Cells

Background

Drugs inhibiting vascular endothelial growth factor (VEGF) signaling are globally administered to suppress deregulated angiogenesis in a variety of eye diseases. However, anti-VEGF therapy potentially affects the normal functions of retinal neurons and glias which constitutively express VEGF receptor 2. Thus, it is desirable to identify novel drug targets which are exclusively expressed in endothelial cells (ECs). Here we attempted to identify an EC-specific Rho guanine nucleotide exchange factor (GEF) and evaluate its role in retinal angiogenesis.

Methodology/Principal Findings

By exploiting fluorescence-activated cell sorting and microarray analyses in conjunction with in silico bioinformatics analyses, we comprehensively identified endothelial genes in angiogenic retinal vessels of postnatal mice. Of 9 RhoGEFs which were highly expressed in retinal ECs, we show that Arhgef15 acted as an EC-specific GEF to mediate VEGF-induced Cdc42 activation and potentiated RhoJ inactivation, thereby promoting actin polymerization and cell motility. Disruption of the Arhgef15 gene led to delayed extension of vascular networks and subsequent reduction of total vessel areas in postnatal mouse retinas.

Conclusions/Significance

Our study provides information useful to the development of new means of selectively manipulating angiogenesis without affecting homeostasis in un-targeted tissues; not only in eyes but also in various disease settings such as cancer.     

Potential of Ayurgenomics Approach in Complex Trait Research: Leads from a Pilot Study on Rheumatoid Arthritis

Background

Inconsistent results across association studies including Genome-wide association, have posed a major challenge in complex disease genetics. Of the several factors which contribute to this, phenotypic heterogeneity is a serious limitation encountered in modern medicine. On the other hand, Ayurveda, a holistic Indian traditional system of medicine, enables subgrouping of individuals into three major categories namely Vata, Pitta and Kapha, based on their physical and mental constitution, referred to as Prakriti. We hypothesised that conditioning association studies on prior risk, predictable in Ayurveda, will uncover much more variance and potentially open up more predictive health.

Objectives and Methods

Identification of genetic susceptibility markers by combining the prakriti based subgrouping of individuals with genetic analysis tools was attempted in a Rheumatoid arthritis (RA) cohort. Association of 21 markers from commonly implicated inflammatory and oxidative stress pathways was tested using a case-control approach in a total cohort comprising 325 cases and 356 controls and in the three subgroups separately. We also tested few postulates of Ayurveda on the disease characteristics in different prakriti groups using clinico-genetic data.

Results

Inflammatory genes like IL1β (C-C-C haplotype, p = 0.0005, OR = 3.09) and CD40 (rs4810485 allelic, p = 0.04, OR = 2.27) seem to be the determinants in Vata subgroup whereas oxidative stress pathway genes are observed in Pitta (SOD3 rs699473, p = 0.004, OR = 1.83; rs2536512 p = 0.005; OR = 1.88 and PON1 rs662, p = 0.04, OR = 1.53) and Kapha (SOD3 rs2536512, genotypic, p = 0.02, OR = 2.39) subgroups. Fixed effect analysis of the associated markers from CD40, SOD3 and TNFα with genotype X prakriti interaction terms suggests heterogeneity of effects within the subgroups. Further, disease characteristics such as severity was most pronounced in Vata group.

Conclusions

This exploratory study suggests discrete causal pathways for RA etiology in prakriti based subgroups, thereby, validating concepts of prakriti and personalized medicine in Ayurveda. Ayurgenomics approach holds promise for biomarker discovery in complex diseases.     

Pathogenicity of aseptic Bursaphelenchus xylophilus

Pine wilt is a disease of pine (Pinus spp.) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus. However, the pathogenic mechanism of pine wilt disease (PWD) remains unclear. Although the PWN was thought to be the only pathogenic agent associated with this disease, a potential role for bacterial symbionts in the disease process was recently proposed. Studies have indicated that aseptic PWNs do not cause PWD in aseptic pine trees, while PWNs associated with bacteria cause wilting symptoms. To investigate the pathogenicity of the PWN and its associated bacteria, 3-month-old microcuttings derived from certain clones of Pinus densiflora Siebold & Zucc. produced in vitro were inoculated under aseptic conditions with aseptic PWNs, non-aseptic PWNs and bacteria isolated from the nematodes. Six-month-old aseptic P. densiflora microcuttings and 7-month-old P. massoniana seedlings were also inoculated under aseptic conditions with aseptic PWNs and non-aseptic PWNs. The results showed that the aseptic microcuttings and seedlings inoculated with aseptic PWNs or non-aseptic PWNs wilted, while those inoculated with bacterial isolates did not wilt. Nematodes were recovered from wilted microcuttings and seedlings inoculated with aseptic PWNs and non-aseptic PWNs, and the asepsis of nematodes recovered from aseptic PWN-inoculated microcuttings and seedlings was reconfirmed by culturing them in NB liquid medium at 30°C for more than 7 days. Taken together, the results indicate that the asepsis of PWN did not cause the loss of pathogenicity.     

A simple and convenient synthesis of 2-methoxyestradiol from estrone

A simple and straightforward synthesis of 2-methoxyestradiol have been achieved in nine synthetic steps with 21% of overall yield. Being a convenient process, it can be upscaled to industrial process.     

Effects of bulkiness and hydrophobicity of an aliphatic amino acid in the recognition helix of the GAGA zinc finger on the stability of the hydrophobic core and DNA binding affinity

The GAGA factor of Drosophila melanogaster uses a single Cys 2His 2-type zinc finger for specific DNA binding. The conformation and DNA binding mode of the GAGA zinc finger are similar to those of other structurally characterized zinc fingers. In almost all Cys 2His 2-type zinc fingers, the fourth position of the DNA-recognizing helix is occupied by the Leu residue involved in the formation of the minimal hydrophobic core. However, no systematic study on the precise role of the Leu residue in the hydrophobic core formation and DNA binding function has been reported. In this study, the Leu residue is substituted with other aliphatic amino acids having different side chain lengths and hydrophobicities, namely, Ile, Val, Aib, and Ala. The metal binding properties were studied by UV-vis spectroscopy. The peptide conformations were examined by CD and NMR spectroscopies. Furthermore, the DNA binding ability was examined with a gel mobility shift assay. Though the Ile, Val, and Aib mutants exhibited conformations similar to those of the wild type, the DNA binding affinity decreased as the side chain length of the amino acid decreased. Interestingly, the Val mutant can bind to the cognate DNA, while Aib cannot, in spite of the similarity in their secondary structures based on the CD measurements. Variable-temperature NMR experiments clearly indicated differences in the stability of the hydrophobic core between the Val and Aib mutants. This study demonstrates that the bulkiness of the conserved aliphatic residue is important in the formation of the well-packed minimal hydrophobic core and proper ternary structure and that the hydrophobic core stabilization is apparently related to the DNA binding function of the GAGA zinc finger.     

Effects of ovariectomy and chronic estradiol administration on pituitary-thyroid axis in adult rats

The effects of ovariectomy (Ovx) and of ovariectomy followed by chronic estradiol dipropionate administration (Ovx EDP) on the structure and function of the pituitary-thyroid axis were examined in the rat. Pituitary TSH cells and thyroid tissue were histologically, immunohistochemically and stereologically investigated. Serum TSH and T(4) levels were determined by RIA. Ovx did not affect pituitary weight, but subsequent treatment with EDP led to its more than two-fold increase (p<0.05). After ovariectomy, the cellular volume of pituitary TSH-immunoreactive cells increased by 28%, p<0.05 compared to sham-operated animals (SO). Treatment of Ovx rats with EDP partially reversed this change. However, the relative volume density of thyrotrophs decreased in comparison to the Ovx and SO groups (by 18% and 23%, p<0.05, respectively). No statistically significant differences in serum TSH levels were observed between the experimental groups. In thyroid tissue both peripheral and central follicles responded to Ovx and EDP treatments. Compared to SO rats, the relative volume densities of the follicles and colloid were increased (by 14% and 30%, p<0.05, respectively) in Ovx rats. Chronic EDP treatment of Ovx rats reversed these changes to the pre-ovariectomy state. Hyperplasia of thyroid follicular cells and a significant reduction (by 21%, p<0.05) of the serum level of T(4) were detected. In conclusion, estradiol deficiency and chronic treatment affected pituitary TSH cells and thyroid tissue. The sum effect of Ovx on the pituitary-thyroid axis was slightly stimulatory. Subsequent EDP treatment decreased thyroid functioning but at the same time preserved serum TSH at the control level.     

A simple, convenient and chemoselective formylation of sterols by Vilsmeier reagent

Vilsmeier reagent (DMF-POCl(3)) was used as an efficient formylating agent. Several sterols having sec-hydroxyl group at 3/17-position have been modified into respective formate esters. The method is simple, mild, chemoselective and provides sec-alcoholic protection in good yields.     

Gallic acid-based indanone derivatives as anticancer agents

Gallic acid-based indanone derivatives have been synthesised. Some of the indanones showed very good anticancer activity in MTT assay. Compounds 10, 11, 12 and 14 possessed potent anticancer activity against various human cancer cell lines. The most potent indanone (10, IC₅₀ = 2.2 μM), against MCF-7, that is, hormone-dependent breast cancer cell line, showed no toxicity to human erythrocytes even at higher concentrations (100 μg/ml, 258 μM). While, indanones 11, 12 and 14 showed toxicities to erythrocytes at higher concentrations.     

Expression of a mutant form of Leishmania donovani centrin reduces the growth of the parasite.

Leishmania donovani, a protozoan parasite, causes visceral disease in humans. To identify genes that control growth, we have isolated for the first time in the order Kinetoplastida a gene encoding for centrin from L. donovani. Centrin is a calcium-binding cytoskeletal protein essential for centrosome duplication or segregation. Protein sequence similarity and immunoreactivity confirmed that Leishmania centrin is a homolog of human centrin 2. Immunofluorescence analysis localized the protein in the basal body. Calcium binding analysis revealed that its C-terminal Ca(2+) binding domain binds 16-fold more calcium than the N-terminal domain. Electrophoretic mobility shift of centrin treated with EGTA and abrogation of the shift in its mutants lacking a Ca(2+) binding site suggest that Ca(2+) binding to these regions may have a role in the protein conformation. The levels of centrin mRNA and protein were high during the exponential growth of the parasite in culture and declined to a low level in the stationary phase. Expression of N-terminal-deleted centrin in the parasite significantly reduces its growth rate, and it was found that significantly more cells are arrested in the G(2)/M stage than in control cells. These studies indicate that centrin may have a functional role in Leishmania growth.