Structure of Smad1 MH1/DNA complex reveals distinctive rearrangements of BMP and TGF-β effectors

Smad1 is a downstream effector of the BMP signaling pathway that binds regulatory DNA to execute gene expression programs leading to, for example, the maintenance of pluripotency in mice. On the contrary, the TGF-β-activated Smad3 triggers strikingly different programs such as mesodermal differentiation in early development. Because Smad1 and Smad3 contain identical amino acids at the DNA contact interface it is unclear how they elicit distinctive bioactivities. Here, we report the crystal structure of the MH1 domain of Smad1 bound to a palindromic Smad binding element. Surprisingly, the DNA contact interface of Smad1 is drastically rearranged when compared to Smad3. The N-terminal helix 1 of Smad1 is dislodged from its intramolecular binding site and adopts a domain swapped arrangement with a symmetry-related molecule. As a consequence, helix 2 kinks away from the double helix disabling several key phosphate backbone interactions. Thermal melting analysis corroborates a decompacted conformation of Smad1 and DNA binding assays indicate a lower overall affinity of Smad1 to DNA but increased cooperativity when binding to palindromic DNA motifs. These findings suggest that Smad1 and Smad3 evolved differential qualities to assemble on composite DNA elements and to engage in co-factor interactions by remodeling their N-termini.     

Surgical removal of duct occluder device under mild hypothermia without cardiopulmonary bypass

Because the use of percutaneous intervention is increasing for the closure of the patent ductus arteriosus, the procedure-related complications are also on rise, with migration of the device being most common. The routine practice is to remove the migrated duct occluder device under cardiopulmonary bypass. Amplatzer duct occluder used in a 4-month-old infant dislodged into the descending thoracic aorta. It was removed by the posterolateral thoracotomy under mild hypothermia through juxtaductal aortotomy between the aortic cross-clamps. The use of cardiopulmonary bypass is thus avoided.     

<Emphasis Type="Italic">Trigonella foenum-graecum</Emphasis> (Fenugreek) Protects Against Selenite-Induced Oxidative Stress in Experimental Cataractogenesis

Cataract is the opacification in eye lens and leads to 50% of blindness worldwide. The present study was undertaken to evaluate the anticataract potential of Trigonella foenum-graecum Linn seeds (fenugreek) in selenite-induced in vitro and in vivo cataract. In vitro enucleated rat lenses were maintained in organ culture containing Dulbecco’s modified Eagles medium (DMEM) alone or in addition with 100 μM selenite and served as the normal and control groups, respectively. For the test group, the medium was supplemented with selenite and T. foenum-graecum aqueous extract. The lenses were incubated for 24 h at 37°C. After incubation, the lenses were processed for the estimation of reduced glutathione (GSH), lipid peroxidation product (malondialdehyde), and the antioxidant enzymes. In vivo selenite cataract was induced in 9-day-old rats by subcutaneous injection of sodium selenite (25 μmol/kg body weight). Animals in the test group were injected with different doses of aqueous extract of T. foenum-graecum 4 h before the selenite challenge. A fall in GSH and a rise in malondialdehyde levels were observed in control as compared to normal lenses. T. foenum-graecum significantly (P < 0.01) restored glutathione and decreased malondialdehyde levels. A significant restoration in the activities of antioxidant enzymes such as superoxide dismutase (P < 0.01), catalase, (P < 0.01), glutathione peroxidase (P < 0.01), and glutathione-S-transferase (P < 0.01) was observed in the T. foenum-graecum supplemented group as compared to control. In vivo, none of the eyes was found with nuclear cataract in treated group as opposed to 72.5% in the control group. T. foenum-graecum protects against experimental cataract by virtue of its antioxidant properties. Further studies are warranted to explore its role in human cataract.     

A multiattribute utility evaluation of different methods for the detection of enteric protozoa causing diarrhea in AIDS patients

Background  

Enteric protozoa and sporozoa have emerged as important opportunistic parasites and can cause fatal infections in AIDS patients. The line of treatment being different for them necessitates an accurate and prompt identification of these to avoid empirical treatment. In this study which is the first of its kind from India we did a comprehensive evaluation of different techniques, comparing them on the basis of the attributes like yield, cost, time taken, expertise and infrastructure. For the first time combination of Calcoflour White and DAPI, a nuclear stain, were used to identify Microsporidia spp. Thus, a diagnostic protocol was devised for rapid, sensitive and cost effective identification of the opportunistic enteric protozoa.     

Effect of sequence hydrophobicity and bilayer width upon the minimum length required for the formation of transmembrane helices in membranes

The minimum hydrophobic length necessary to form a transmembrane (TM) helix in membranes was investigated using model membrane-inserted hydrophobic helices. The fluorescence of a Trp at the center of the sequence and its sensitivity to quenching were used to ascertain helix position within the membrane. Peptides with hydrophobic cores composed of poly(Leu) were compared to sequences containing a poly 1:1 Leu:Ala core (which have a hydrophobicity typical of natural TM helices). Studies varying bilayer width revealed that the poly(Leu) core peptides predominately formed a TM state when the bilayer width exceeded hydrophobic sequence length by (i.e. when negative mismatch was) up to ∼ 11-12 Å (e.g. the case of a 11-12 residue hydrophobic sequence in bilayers with a biologically relevant width, i.e. dioleoylphosphatidylcholine (DOPC) bilayers), while poly(LeuAla) core peptides formed predominantly TM state with negative mismatch of up to 9 Å (a 13 residue hydrophobic sequence in DOPC bilayers). This indicates that minimum length necessary to form a predominating amount of a TM state (minimum TM length) is only modestly hydrophobicity-dependent for the sequences studied here, and a formula that defines the minimum TM length as a function of hydrophobicity for moderately-to-highly hydrophobic sequences was derived. The minimum length able to form a stable TM helix for alternating LeuAla sequences, and that for sequences with a Leu block followed by an Ala block, was similar, suggesting that a hydrophobicity gradient along the sequence may not be an important factor in TM stability. TM stability was also similar for sequences flanked by different charged ionizable residues (Lys, His, Asp). However, ionizable flanking residues destabilized the TM configuration much more when charged than when uncharged. The ability of short hydrophobic sequences to form TM helices in membranes in the presence of substantial negative mismatch implies that lipid bilayers have a considerable ability to adjust to negative mismatch, and that short TM helices may be more common than generally believed. Factors that modulate the ability of bilayers to adjust to mismatch may strongly affect the configuration of short hydrophobic helices.     

RAD51C deficiency in mice results in early prophase I arrest in males and sister chromatid separation at metaphase II in females

RAD51C is a member of the RecA/RAD51 protein family, which is known to play an important role in DNA repair by homologous recombination. In mice, it is essential for viability. Therefore, we have generated a hypomorphic allele of Rad51c in addition to a null allele. A subset of mice expressing the hypomorphic allele is infertile. This infertility is caused by sexually dimorphic defects in meiotic recombination, revealing its two distinct functions. Spermatocytes undergo a developmental arrest during the early stages of meiotic prophase I, providing evidence for the role of RAD51C in early stages of RAD51-mediated recombination. In contrast, oocytes can progress normally to metaphase I after superovulation but display precocious separation of sister chromatids, aneuploidy, and broken chromosomes at metaphase II. These defects suggest a possible late role of RAD51C in meiotic recombination. Based on the marked reduction in Holliday junction (HJ) resolution activity in Rad51c-null mouse embryonic fibroblasts, we propose that this late function may be associated with HJ resolution.     

Luminescence studies of perturbation of tryptophan residues of tubulin in the complexes of tubulin with colchicine and colchicine analogues

Tubulin, a heterodimeric (alphabeta) protein, the main constituent of microtubules, binds efficiently with colchicine (consisting of a trimethoxybenzene ring, a seven-member ring and methoxy tropone moiety) and its analogues, viz., demecolcine and AC [2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone]. Tubulin contains eight tryptophan (Trp) residues at A21, A346, A388, A407, B21, B103, B346, and B407 in the two subunits. The role of these eight Trp residues in this interaction and also their perturbation due to binding have been explored via time-resolved fluorescence at room temperature and low-temperature (77 K) phosphorescence in a suitable cryosolvent. Both the time-resolved fluorescence data and 77 K phosphorescence spectra indicate that the emitting residues move toward a more hydrophobic and less polar environment after complex formation. The environment of emitting Trps in the complex also becomes slightly more heterogeneous. Our analysis using the experimental results, the calculation of the accessible surface area (ASA) of all the Trps in the wild type and tubulin-colchicine complex [Ravelli, R. B. G., et al. (2004) Nature 428, 198-202], the distance of the Trp residues from the different moieties of the colchicine molecule, the knowledge of the nature of the immediate residues (<5 A) present near each Trp residue, and the calculation of the intramolecular Trp-Trp energy transfer efficiencies indicate that Trp A346, Trp A407, Trp B21, and Trp B407 are the major contributors to the emission in the free protein, while Trp B21 and Trp B103 are mainly responsible for the emission of the complexes. A comparative account of the photophysical aspects of the drug molecules bound to protein in aqueous buffer and in buffer containing 40% ethylene glycol has been presented. The quantum yield and average lifetime of fluorescence in tubulin and its complexes with colchicine are used to predict the possible donors and the energy transfer (ET) efficiency in the ET process from Trps to colchicine in the complex. This study is a unique attempt to identify the Trp residues contributing to the emission in the free protein and in a complex of a multi-Trp protein with a drug molecule without performing the mutation of the protein.     

Synthesis and in vitro antibacterial activity of novel methylamino piperidinyl oxazolidinones

Design and synthesis of a few novel methylamino piperidinyl substituted oxazolidinones are reported. Their antibacterial activities have been evaluated in a MIC assay against broader panel of both susceptible and resistant Gram-positive strains. (S)-N-{3-[3-Fluoro-4-(methyl-{1-[3-(5-nitrofuran-2-yl)-acryloyl]-piperidin-4-yl}-amino)-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide 4i has shown comparable antibacterial activity to linezolid and eperezolid in the MIC assay, additionally compound 4i showed good antibacterial activity with an in vitro MIC value of 2-4 microg/mL against linezolid resistant Staphylococcus aureus (linezolid 16 microg/mL).     

Ultrasensitive detection of cortisol with enzyme fragment complementation technology using functionalized nanowire

Cortisol is a member of the glucocorticoid hormone family and a key metabolic regulator. Increased intracellular cortisol levels have been implicated in type 2 diabetes, obesity, and metabolic syndrome. Cortisol is an important bio-marker of stress and its detection is also important in sports medicine. However, rapid methods for sensitive detection of cortisol are limited. Functionalized gold nanowires were used to enhance the sensitivity and selectivity of cortisol detection. Gold nanowires are used to improve the electron transfer between the electrodes. Moreover, the large surface to volume ratio, small diffusion time and high electrical conductivity and their aligned nature will enhance the sensitivity and detection limit of the biosensor several fold. The biosensor was fabricated using, aligned gold (Au) nanowires to behave as the working electrode, platinum deposited on a silicon chip to function as the counter electrode, and silver/silver chloride as reference electrode. The gold nanowires were coupled with cortisol antibodies using covalent linkage chemistry and a fixed amount of 3alpha-hydroxysteroid dehydrogenase was introduced into the reaction cell during each measurement to convert (reduce) ketosteroid into hydroxyl steroid. Furthermore, the micro-fluidic, micro-fluid part of the sensor was fabricated using micro-electro-mechanical system (MEMS) technology to have better control on liquid flow over Au nanowires to minimize the signal to noise ratio. The biosensor was characterized using SEM, AFM and FTIR technique. The response curve of the biosensor was found to be linear in the range of 10-80 microM of cortisol. Moreover, the presence of hydrocortisone is sensitively detected in the range of 5-30 microM. It is concluded that the functionalized gold nanowires with micro-fluidic device using enzyme fragment complementation technology can provide an easy and sensitive assay for cortisol detection in serum and other biological fluids.