The Rad27 (Fen-1) nuclease inhibits Ty1 mobility in Saccharomyces cerevisiae

Although most Ty1 elements in Saccharomyces cerevisiae are competent for retrotransposition, host defense genes can inhibit different steps of the Ty1 life cycle. Here, we demonstrate that Rad27, a structure-specific nuclease that plays an important role in DNA replication and genome stability, inhibits Ty1 at a post-translational level. We have examined the effects of various rad27 mutations on Ty1 element retrotransposition and cDNA recombination, termed Ty1 mobility. The point mutations rad27-G67S, rad27-G240D, and rad27-E158D that cause defects in certain enzymatic activities in vitro result in variable increases in Ty1 mobility, ranging from 4- to 22-fold. The C-terminal frameshift mutation rad27-324 confers the maximum increase in Ty1 mobility (198-fold), unincorporated cDNA, and insertion at preferred target sites. The null mutation differs from the other rad27 alleles by increasing the frequency of multimeric Ty1 insertions and cDNA recombination with a genomic element. The rad27 mutants do not markedly alter the levels of Ty1 RNA or the TyA1-gag protein. However, there is an increase in the stability of unincorporated Ty1 cDNA in rad27-324 and the null mutant. Our results suggest that Rad27 inhibits Ty1 mobility by destabilizing unincorporated Ty1 cDNA and preventing the formation of Ty1 multimers.     

SSR and AFLP based genetic diversity of soybean germplasm differing in photoperiod sensitivity

Forty-four soybean genotypes with different photoperiod response were selected after screening of 1000 soybean accessions under artificial condition and were profiled using 40 SSR and 5 AFLP primer pairs. The average polymorphism information content (PIC) for SSR and AFLP marker systems was 0.507 and 0.120, respectively. Clustering of genotypes was done using UPGMA method for SSR and AFLP and correlation was 0.337 and 0.504, respectively. Mantel's correlation coefficients between Jaccard's similarity coefficient and the cophenetic values were fairly high in both the marker systems (SSR = 0.924; AFLP = 0.958) indicating very good fit for the clustering pattern. UPGMA based cluster analysis classified soybean genotypes into four major groups with fairly moderate bootstrap support. These major clusters corresponded with the photoperiod response and place of origin. The results indicate that the photoperiod insensitive genotypes, 11/2/1939 (EC 325097) and MACS 330 would be better choice for broadening the genetic base of soybean for this trait.     

Attenuation of dengue virus infection by adeno-associated virus-mediated siRNA delivery

BACKGROUND: The need for safe and effective treatment of dengue virus (DEN), a class A agent that causes dengue hemorrhagic fever/dengue shock syndrome, has been a critical global priority. An effective vaccine for DEN is not yet available. In this study the possibility of attenuating DEN infection using adeno-associated virus (AAV)-encoded short interfering RNAs (siRNA) was examined in Vero cells and human dendritic cells (DCs). METHODS: A cassette encoding siRNA targeted to a 3' untranslated sequence common to all DEN serotypes was designed and tested for its ability to attenuate DEN infection by use of AAV delivery. RESULTS: Vero cells or DCs infected with AAV-siRNA showed a significant, dose-dependent reduction in DEN infection. Treatment of DCs with AAV-siRNA also decreased the DEN-induced apoptosis of DCs and did not induce significant inflammation. CONCLUSION: These results demonstrate that AAV-mediated siRNA delivery is capable of reducing DEN infection in cells and may be useful in decreasing DEN replication in humans.     

Role of putrescine in ovary and embryo development in fruit bat Cynopterus sphinx during embryonic diapause

The aim of this study was to evaluate the effect of putrescine on ovarian activity and the rate of embryonic development in Cynopterus sphinx during delayed development. The result showed the presence of a rate‐limiting enzyme, ornithine decarboxylase‐1, in both ovary and utero‐embryonic unit of C. sphinx suggests a synthesis of putrescine in these sites. The corpus luteum showed increased, whereas utero‐embryonic unit showed decreased production of putrescine during delayed development as compared with the normal development. The bat treated in vivo with putrescine during delayed development showed increase in progesterone and estradiol synthesis, correlated with increased expression of luteinizing hormone receptor, steroidogenic acute receptor protein, and 3β‐hydroxysteroid dehydrogenase through extracellular signal‐regulated kinase (ERK1/2)‐mediated pathway in the ovary; but showed increase in the weight and expression of progesterone receptor (PR), B‐cell lymphoma 2, proliferating cell nucleus antigen, and vascular endothelial growth factor proteins in utero‐embryonic unit. The in vitro treatment of putrescine showed stimulatory whereas treatment with an inhibitor of putrescine, 2‐difluoromethylornithine caused an inhibitory effect on ovarian progesterone synthesis and cell proliferation, and cell survival in the utero‐embryonic unit. In conclusion, the putrescine showed two separate roles during embryonic diapause, high concentration of putrescine in the ovary may support corpus luteum and basal synthesis of progesterone, whereas a low level of putrescine causes retarded embryonic development by inhibiting cell proliferation in the utero‐embryonic unit. The bat treated with putrescine either directly promotes cell proliferation, cell survival, and angiogenic activities or acts indirectly increasing PR on utero‐embryonic unit thereby activating development in delayed embryo in C. sphinx.     

A prospective observational study to sequentially determine the dermoscopic features of vitiligo and its association with disease activity in patients on medical treatment

Prospective data on correlation between dermoscopic features of vitiligo and disease activity are scarce. This study was conducted to sequentially determine the dermoscopic features of vitiligo and to evaluate their association with disease activity. A cohort of 30 patients with 60 active vitiligo patches on medical therapy was subjected to sequential clinical and dermoscopic examination at four weekly intervals till 16 weeks. Disease activity at each visit was assessed using serial clinical photographs and modified vitiligo activity severity index. The dermoscopic images were merged and analysed for a predefined set of dermoscopic parameters by two blinded dermatologists. Paired analysis of dermoscopic features was done between baseline, and stabilized vitiligo patches at 12 and 16 weeks. Pigment network changes (absent and reduced pigment network, p < .001), perifollicular depigmentation (p = .02), ill-defined margins (p = .04) and satellite lesions and micro-Koebner phenomenon (p < .001) were associated with active vitiligo while perifollicular repigmentation (p < .001) was associated with stabilizing and repigmenting vitiligo. Satellite lesions and micro-Koebner's phenomena were suggestive of unstable disease irrespective of site of target lesion, while perifollicular repigmentation was suggestive of stabilized/repigmenting disease only at non-acral sites. We found sequential dermoscopy to be useful to assess disease activity and potential for repigmentation in localized vitiligo.     

Developmental phase-dependent photosynthetic responses to ultraviolet-B radiation: damage, defence, and adaptation of primary leaves of wheat seedlings

Alterations in photosynthetic capacity of primary leaves of wheat seedlings in response to ultraviolet-B (UV-B; 280–320 nm; 60 μmol m⁻² s⁻¹) exposure alone and in combination with photosynthetically active radiation (PAR; 400–800 nm; 200 μmol m⁻² s⁻¹) during different phases of leaf growth and development were assessed. UV-B exposure resulted in a phase-dependent differential loss in photosynthetic pigments, photochemical potential, photosystem 2 (PS2) quantum yield, and in vivo O₂ evolution. UV-B exposure induced maximum damage to the photosynthetic apparatus during senescence phase of development. The damages were partially alleviated when UV-B exposure was accompanied by PAR. UV-B induced an enhancement in accumulation of flavonoids during all phases of development while it caused a decline in anthocyanin content during senescence. The differential changes in these parameters demonstrated the adaptation ability of leaves to UV-B stress during all phases of development and the ability was modified in UV-B+ PAR exposed samples.     

High-frequency plant regeneration by <Emphasis Type="Italic">in vitro</Emphasis> shoot proliferation in cotyledonary node explants of grasspea (<Emphasis Type="Italic">Lathyrus sativus</Emphasis> L.)

Summary Multiple shoots were induced from cotyledonary nodes of grasspea (Lathyrus sativus L.) derived from 7-d-old in vitro seedlings on Murashige and Skoog (MS) medium containing N⁶-benzyladenine (BA), kinetin, or thidiazuron, BA being the most effective. Among the five genotypes tested, shoot proliferation frequency was the highest (93.3%) for IC-120487, giving the maximum number of shoots (11.3 shoots per explant) on MS medium augmented with 2.0 mgl⁻¹ (8.87 μM) BA. Shoot cultures were established by repeatedly subculturing the original cotyledonary nodes on fresh medium after each harvest of the newly formed shoots. Thus 30–40 shoots were obtained in 2 mo. from a single cotyledonary node. Up to 81.8% of the shoots developed roots following transfer to half-strength MS medium containing 0.5 mgl⁻¹ (2.85 μM) indole-3-acetic acid. Plantlets were successfully acclimatized and established in soil.     

Inhibition of type IA topoisomerase by a monoclonal antibody through perturbation of DNA cleavage-religation equilibrium

Type IA DNA topoisomerases, typically found in bacteria, are essential enzymes that catalyse the DNA relaxation of negative supercoils. DNA gyrase is the only type II topoisomerase that can carry out the opposite reaction (i.e. the introduction of the DNA supercoils). A number of diverse molecules target DNA gyrase. However, inhibitors that arrest the activity of bacterial topoisomerase I at low concentrations remain to be identified. Towards this end, as a proof of principle, monoclonal antibodies that inhibit Mycobacterium smegmatis topoisomerase I have been characterized and the specific inhibition of Mycobacterium smegmatis topoisomerase I by a monoclonal antibody, 2F3G4, at a nanomolar concentration is described. The enzyme-bound monoclonal antibody stimulated the first transesterification reaction leading to enhanced DNA cleavage, without significantly altering the religation activity of the enzyme. The stimulated DNA cleavage resulted in perturbation of the cleavage-religation equilibrium, increasing single-strand nicks and protein-DNA covalent adducts. Monoclonal antibodies with such a mechanism of inhibition can serve as invaluable tools for probing the structure and mechanism of the enzyme, as well as in the design of novel inhibitors that arrest enzyme activity.