Analysis of the impact of a uracil DNA glycosylase attenuated in AP-DNA binding in maintenance of the genomic integrity in Escherichia coli

Uracil DNA glycosylase (Ung)initiates the uracil excision repair pathway. We have earlier characterized the Y66W and Y66H mutants of Ung and shown that they are compromised by similar to 7- and similar to 170-fold, respectively in their uracil excision activities. In this study, fluorescence anisotropy measurements show that compared with the wild-type, the Y66W protein is moderately compromised and attenuated in binding to AP-DNA. Allelic exchange of ung in Escherichia coli with ung::kan, ungY66H:amp or ungY66W:amp alleles showed similar to 5-, similar to 3.0- and similar to 2.0-fold, respectively increase in mutation frequencies. Analysis of mutations in the rifampicin resistance determining region of rpoB revealed that the Y66W allele resulted in an increase in A to G (or T to C) mutations. However, the increase in A to G mutations was mitigated upon expression of wild-type Ung from a plasmid borne gene. Biochemical and computational analyses showed that the Y66W mutant maintains strict specificity for uracil excision from DNA. Interestingly, a strain deficient in AP-endonucleases also showed an increase in A to G mutations. We discuss these findings in the context of a proposal that the residency of DNA glycosylase(s) onto the AP-sites they generate shields them until recruitment of AP-endonucleases for further repair.

Cationic Amino Acid Transporters and Salmonella Typhimurium ArgT Collectively Regulate Arginine Availability towards Intracellular Salmonella Growth

Cationic amino acid transporters (mCAT1 and mCAT2B) regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV) specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells.

Recombinant mid gut antigen (Bm95) as a vaccine against Indian Rhiphicephalus haemaphysaloides in Bos indicus cattle

In the present study, we report for the first time the efficacy of recombinant Bm95 mid gut antigen isolated from an Argentinean strain of Rhipicephalus microplus strain A in controlling the tick infestations in India. The synthetic gene for Bm95 optimized for expression in yeast was obtained and used to generate yeast transformants expressing Bm95 which was purified to apparent homogeneity. Liquid chromatography-mass spectrometry analysis of the purified protein confirmed its identity as Bm95. Vaccine was prepared by blending various concentrations of purified Bm95 with aluminium hydroxide as an adjuvant. lmmunogenicity studies of the vaccine in rabbits and cattle indicated that the vaccine was highly immunogenic. The efficacy studies of the vaccine was done in cattle. Naive Bos indicus cattle were vaccinated with the recombinant vaccine and were challenged with the larval, nymphal and adult forms of Rhiphicephalus haemaphysaloides. The vaccine protected the animals from larval, nymph and adult tick challenges with an efficacy of 98.7%, 84.6% and 78.9% respectively. The results obtained from the above studies clearly demonstrated the advantage and possibilities of the use of Bm95 in controlling R. haemaphysaloides infestations in the field. (C) 2010 Elsevier Ltd. All rights reserved.

Calcium/Calmodulin Dependent Protein Kinase II Bound to NMDA Receptor 2B Subunit Exhibits Increased ATP Affinity and Attenuated Dephosphorylation

Calcium/calmodulin dependent protein kinase II (CaMKII) is implicated to play a key role in learning and memory. NR2B subunit of N-methyl-D-aspartate receptor (NMDAR) is a high affinity binding partner of CaMKII at the postsynaptic membrane. NR2B binds to the T-site of CaMKII and modulates its catalysis. By direct measurement using isothermal titration calorimetry (ITC), we show that NR2B binding causes about 11 fold increase in the affinity of CaMKII for ATP gamma S, an analogue of ATP. ITC data is also consistent with an ordered binding mechanism for CaMKII with ATP binding the catalytic site first followed by peptide substrate. We also show that dephosphorylation of phospho-Thr(286)-alpha-CaMKII is attenuated when NR2B is bound to CaMKII. This favors the persistence of Thr(286) autophosphorylated state of CaMKII in a CaMKII/phosphatase conjugate system in vitro. Overall our data indicate that the NR2B- bound state of CaMKII attains unique biochemical properties which could help in the efficient functioning of the proposed molecular switch supporting synaptic memory.

Needleless Vaccine Delivery Using Micro-Shock Waves

Shock waves are one of the most efficient mechanisms of energy dissipation observed in nature. In this study, utilizing the instantaneous mechanical impulse generated behind a micro-shock wave during a controlled explosion, a novel nonintrusive needleless vaccine delivery system has been developed. It is well-known that antigens in the epidermis are efficiently presented by resident Langerhans cells, eliciting the requisite immune response, making them a good target for vaccine delivery. Unfortunately, needle-free devices for epidermal delivery have inherent problems from the perspective of the safety and comfort of the patient. The penetration depth of less than 100 mu m in the skin can elicit higher immune response without any pain. Here we show the efficient utilization of our needleless device (that uses micro-shock waves) for vaccination. The production of liquid jet was confirmed by high-speed microscopy, and the penetration in acrylamide gel and mouse skin was observed by confocal microscopy. Salmonella enterica serovar Typhimurium vaccine strain pmrG-HM-D (DV-STM-07) was delivered using our device in the murine salmonellosis model, and the effectiveness of the delivery system for vaccination was compared with other routes of vaccination. Vaccination using our device elicits better protection and an IgG response even at a lower vaccine dose (10-fold less) compared to other routes of vaccination. We anticipate that our novel method can be utilized for effective, cheap, and safe vaccination in the near future.

A novel zinc bis(thiosemicarbazone) complex for live cell imaging

Fluorescent zinc complexes have recently attracted a lot of interest owing to their vast applications in cellular imaging. We report the synthesis as well as physical, chemical and biological studies of a novel zinc glyoxalbis(4-methyl-4-phenyl-3-thiosemicarbazone), Zn (GTSC)](3), complex. As compared with the well-studied zinc biacetylbis(4-methyl-3-thiosemicarbazone), Zn(ATSM), complex, which was used as a reference, Zn(GTSC)](3) had 2.5-fold higher fluorescence. When cellular fluorescence was measured using flow cytometry, we observed that Zn(GTSC)](3) had 3.4-fold to 12-fold higher fluorescence than Zn(ATSM) in various cell lines (n = 9) of different tissue origin. Confocal fluorescence microscopy results showed that Zn(GTSC)](3) appeared to have a nuclear localization within 30 mm of addition to MCF7 cells. Moreover, Zn(GTSC)](3) showed minimal cytotoxicity compared with Zn(ATSM), suggesting that Zn(GTSC)](3) may be less deleterious to cells when used as an imaging agent. Our data suggest that the novel Zn(GTSC)](3) complex can potentially serve as a biocompatible fluorescent imaging agent for live cells.

Responsiveness of human male volunteers to immunization with ovine follicle stimulating hormone vaccine: Results of a pilot study

A study of 140 days duration was performed to examine if human male volunteers (n = 5) respond to ovine follicle stimulating hormone (oFSH) immunization (administered adsorbed on Alugel on days 1, 20, 40 and 70) by producing antibodies capable of both binding and neutralizing bioactivity of human FSH. The kinetics of antibody production for both the immunogen (oFSH) and the cross-reactive antigen (hFSH) were essentially similar, The volunteers responded only to the first two immunizations, The boosters given on days 40 and 70 were ineffective, probably because of the presence of substantial amounts of circulating antibody to oFSH. Of the antibodies generated to oFSH, 25-45% bound hFSH with a mean binding affinity of 0.65 x 10(9) +/- 0.53 M(-1). The binding capacities at the time of high (30-80 days of immunization) and low (>110 days) titres were 346 +/- 185 and 10.5 +/- 5.8 ng hFSH/ml respectively, During the period of high titre, free serum FSH (value in normal males 1-5 ng/ml) was not monitorable, A 50 mu l aliquot of the antiserum obtained from different volunteers between days 30 and 80 and on day 140 blocked binding of I-125-labelled hFSH to its receptor by 82 +/- 9.7 and 53 +/- 12.2% respectively, The antibody produced was specific for FSH, and no significant change in the values of related glycoprotein hormones (luteinizing hormone/testosterone and thyroid stimulating hormone/thyroxine) were recorded, Seminal plasma transferrin, a marker of Sertoli cell as well as of seminiferous tubular function, showed marked reduction (30-90%) following immunization with oFSH. Considering that endogenous FSH remained neutralized for approximately one sperm cycle only (65 days), the reduction in sperm counts (30-74%) exhibited by some volunteers is encouraging, Immunization with oFSH did not result in any significant changes in haematology, serum biochemistry or hormonal profiles, There was no production of antibodies capable of interacting with non-specific tissues, It is concluded that it should be possible to obtain a sustained long-term blockade of endogenous FSH action in men by using oFSH as an immunogen, This is a prerequisite for obtaining significant reduction in the quality and quantity of spermatozoa produced, thus leading to infertility.

A phenanthrene based highly selective fluorogenic and visual sensor for Cu2+ ion with nanomolar detection limit and its application in live cell imaging

A new phenanthrene based chemosensor has been synthesized and investigated to act as highly selective fluorescence and visual sensor for Cu2+ ion with very low detection limit of 1.58 nM: this has also been used to image Cu2+ in human cervical HeLa cancer cells. (C) 2012 Elsevier B.V. All rights reserved.

Live Soap: Stability, Order, and Fluctuations in Apolar Active Smectics

We construct a hydrodynamic theory of noisy, apolar active smectics in bulk suspension or on a substrate. Unlike purely orientationally ordered active fluids, active apolar smectics can be dynamically stable in Stokesian bulk suspensions. Smectic order in these systems is quasilong ranged in dimension d = 2 and long ranged in d = 3. We predict reentrant Kosterlitz-Thouless melting to an active nematic in our simplest model in d = 2, a nonzero second-sound speed parallel to the layers in bulk suspensions, and that there are no giant number fluctuations in either case. We also briefly discuss possible instabilities in these systems. DOI: 10.1103/PhysRevLett.110.118102

Growth kinetics and immune response of chimeric foot-and-mouth disease virus serotype `O' produced through replication competent mini genome of serotype Asia 1, 63/72, in BHK cell lines

Regular vaccinations with potent vaccine, in endemic countries and vaccination to live in non-endemic countries are the methods available to control foot-and-mouth disease. Selection of candidate vaccine strain is not only cumbersome but the candidate should grow well for high potency vaccine preparation. Alternative strategy is to generate an infectious cDNA of a cell culture-adapted virus and use the replicon for development of tailor-made vaccines. We produced a chimeric `O' virus in the backbone of Asia 1 and studied its characteristics. The chimeric virus showed high infectivity titre (>10(10)) in BHK 21 cell lines, revealed small plague morphology and there was no cross reactivity with antiserum against Asia I. The virus multiplies rapidly and reaches peak at 12 h post infection. The vaccine prepared with this virus elicited high antibody titres.

Image deconvolution research: its scope and importance in live cell microscopy

Fluorescence microscopy has become an indispensable tool in cell biology research due its exceptional specificity and ability to visualize subcellular structures with high contrast. It has highest impact when applied in 4D mode, i.e. when applied to record 3D image information as a function of time, since it allows the study of dynamic cellular processes in their native environment. The main issue in 4D fluorescence microscopy is that the phototoxic effect of fluorescence excitation gets accumulated during 4D image acquisition to the extent that normal cell functions are altered. Hence to avoid the alteration of normal cell functioning, it is required to minimize the excitation dose used for individual 2D images constituting a 4D image. Consequently, the noise level becomes very high degrading the resolution. According to the current status of technology, there is a minimum required excitation dose to ensure a resolution that is adequate for biological investigations. This minimum is sufficient to damage light-sensitive cells such as yeast if 4D imaging is performed for an extended period of time, for example, imaging for a complete cell cycle. Nevertheless, our recently developed deconvolution method resolves this conflict forming an enabling technology for visualization of dynamical processes of light-sensitive cells for durations longer than ever without perturbing normal cell functioning. The main goal of this article is to emphasize that there are still possibilities for enabling newer kinds of experiment in cell biology research involving even longer 4D imaging, by only improving deconvolution methods without any new optical technologies.