Numblike and Numb differentially affect p53 and Sonic Hedgehog signaling

Numb serves as a key regulator of Notch and Sonic Hedgehog signaling and also modulates p53 protein levels. Numblike is a highly conserved homolog to mammalian Numb, but considerably less is known about its function. To address the role of Numblike, we have generated a mouse embryonic stem (ES) cell line, Nbl^TetOn, in which expression of Numblike can be induced and analyzed the effect of activation of Numblike. Induction of Numblike, similar to Numb, reduced the amount of Notch receptor, whereas Numblike differed from Numb with regard to p53 and Shh signaling. In contrast to Numb, Numblike did not elevate the level of p53 protein and Numblike potentiated, rather than reduced, the immediate downstream response of Shh signaling. In keeping with a role for Numblike in potentiating Shh signaling, Shh and Numblike synergistically increased the proportion of ES cells expressing pluripotency markers. In conclusion, the data demonstrate that Numb and Numblike have evolved to acquire at least partially distinct functions.     

Local and global dynamics of the G protein-coupled receptor CXCR1

The local and global dynamics of the chemokine receptor CXCR1 are characterized using a combination of solution NMR and solid-state NMR experiments. In isotropic bicelles (q = 0.1), only 13% of the expected number of backbone amide resonances is observed in (1)H/(15)N HSQC solution NMR spectra of uniformly (15)N-labeled samples; extensive deuteration and the use of TROSY made little difference in the 800 MHz spectra. The limited number of observed amide signals is ascribed to mobile backbone sites and assigned to specific residues in the protein; 19 of the signals are from residues at the N-terminus and 25 from residues at the C-terminus. The solution NMR spectra display no evidence of local backbone motions from residues in the transmembrane helices or interhelical loops of CXCR1. This finding is reinforced by comparisons of solid-state NMR spectra of both magnetically aligned and unoriented bilayers containing either full-length or doubly N- and C-terminal truncated CXCR1 constructs. CXCR1 undergoes rapid rotational diffusion about the normal of liquid crystalline phospholipid bilayers; reductions in the frequency span and a change to axial symmetry are observed for both carbonyl carbon and amide nitrogen chemical shift powder patterns of unoriented samples containing (13)C- and (15)N-labeled CXCR1. In contrast, when the phospholipids are in the gel phase, CXCR1 does not undergo rapid global reorientation on the 10(4) Hz time scale defined by the carbonyl carbon and amide nitrogen chemical shift powder patterns.     

(5'S)-8,5'-cyclo-2'-deoxyguanosine is a strong block to replication, a potent pol V-dependent mutagenic lesion, and is inefficiently repaired in Escherichia coli

8,5'-Cyclopurines, making up an important class of ionizing radiation-induced tandem DNA damage, are repaired only by nucleotide excision repair (NER). They accumulate in NER-impaired cells, as in Cockayne syndrome group B and certain Xeroderma Pigmentosum patients. A plasmid containing (5'S)-8,5'-cyclo-2'-deoxyguanosine (S-cdG) was replicated in Escherichia coli with specific DNA polymerase knockouts. Viability was <1% in the wild-type strain, which increased to 5.5% with SOS. Viability decreased further in a pol II(-) strain, whereas it increased considerably in a pol IV(-) strain. Remarkably, no progeny was recovered from a pol V(-) strain, indicating that pol V is absolutely required for bypassing S-cdG. Progeny analyses indicated that S-cdG is significantly mutagenic, inducing ~34% mutation with SOS. Most mutations were S-cdG → A mutations, though S-cdG → T mutation and deletion of 5'C also occurred. Incisions of purified UvrABC nuclease on S-cdG, S-cdA, and C8-dG-AP on a duplex 51-mer showed that the incision rates are C8-dG-AP > S-cdA > S-cdG. In summary, S-cdG is a major block to DNA replication, highly mutagenic, and repaired slowly in E. coli.     

Sorbitan ester niosomes for topical delivery of rofecoxib

The aim of the present investigation is to encapsulate rofecoxib in niosomes and incorporate the prepared niosomes into dermal gel base for sustained therapeutic action. Niosomes were prepared by lipid film hydration technique and were analyzed for size, entrapment efficiency and drug retention capacity. Niosomal vesicles were then incorporated into blank carbopol gel to form niosomal gel. The in vitro permeation study across pig skin was performed using Keshary-Chien glass diffusion cell. The size and entrapment efficiency of the niosomal vesicles increased with gradual increase in HLB value of nonionic surfactants used. Maximum drug entrapment was observed with Span 20 with HLB value of 8.6 and drug leakage from vesicles was less at refrigerated condition than at the room temperature. Higher proportion of cholesterol made the niosomal formulation more stable with high drug retention properties. The niosomal gel showed a prolong drug release behavior compared to plain drug gel. Differential scanning calorimetric study of drug loaded gel and pig skin after permeation study confirmed inertness of carbopol gel base toward rofecoxib and absence of drug metabolism in the skin during permeation study, respectively. The niosomal formulations were successfully prepared by lipid film hydration technique using cholesterol and Span as nonionic surfactant. Presence of cholesterol made niosomes more stable with high drug entrapment efficiency and retention properties. The lower flux value of niosomal gel as compared to plain drug gel across pig skin assured the prolong drug release behavior with sustained action.     

Comparative study of 6-APA production by free and agar immobilized bacteria in nutrient broth culture

In the present study different bacterial samples were isolated from soil of different places of Dibrugarh and screened for biotransformation ability to produce 6-Aminopenicillanic acid. Among ten isolated bacterial samples, three gram positive bacterial samples designated as AKDD-2, AKDD-4 and AKDD-6 showed the production of 6-APA from penicillin G. Assessment of production of 6-APA after incubation in penicillin G (2 mg/ml) by three different samples separately in free and agar immobilization state was done by HPLC analysis. Reusability of immobilized cells was found successful up to 14 days.     

Synthesis and characterisation of ammonium mediated assembly of two neutral nickel(II) Schiff base fragments

The 1:2 condensation of o-phenelenediamine and o-vanilline yields a compartmental N₂O₄ ligand N,N′-(1,2-Phenylene)-bis(3-methoxysalicylideneimine) [H₂L]. When nickel(II) thiocyanate is added to the methanol solution of H₂L, followed by addition of ammonium thiocyanate, an unusual nickel(II) compound, [NH₄(NiL)₂SCN]·H₂O (1), is separated out in which an ammonium ion is sandwiched between two neutral square planner NiL moieties. Hydrogen bonding interactions are observed among the ammonium ion, NiL moieties, the thiocyanate anion and the water of crystallization. The compound is characterized by C, H, N analysis, UV-VIS and IR spectroscopy, room temperature magnetic susceptibility measurement and X-ray crystal diffraction study. The compound crystallizes in monoclinic space group P2_1/n with a = 13.8636(7) Å, b = 14.0267(7) Å, c = 22.2715(10) Å and β = 94.301(3)°.     

Population genetic analyses of Plasmodium falciparum chloroquine receptor transporter gene haplotypes reveal the evolutionary history of chloroquine-resistant malaria in India

Inferring the origin and dispersal of the chloroquine-resistant (CQR) malaria parasite, Plasmodium falciparum, is of academic and public health importance. The Pfcrt gene of P. falciparum is widely known as the CQR gene and two major haplotypes of this gene (CVIET and SVMNT) occur widely across CQR-endemic regions of the globe. In India, studies to date of the Pfcrt gene have indicated the widespread prevalence of the SVMNT haplotype (prevalent in the South America and Papua New Guinea), whereas the CVIET haplotype, primarily found in southeast Asia, was not detected at a high frequency in India. This distribution pattern of the two most common CQR-Pfcrt haplotypes in India is quite surprising. Thus, in order to understand probable evolutionary and migration patterns of the CQR-Pfcrt haplotypes into India, we generated new sequence data of exon 2 of the Pfcrt gene and collected published information on the CQR-Pfcrt haplotype data from India, Papua New Guinea, southeast Asia and South America, and performed several population and evolutionary genetic analyses. Among several interesting findings, statistically significant longitudinal clines for the CVIET and SVMNT haplotypes (in opposite directions) in India, and the clustering of India and Papua New Guinea under the SVMNT-specific clade in the phylogenetic tree, are the two most remarkable aspects of the data. It also appears that both the SVMNT and CVIET haplotypes in India have migrated from southeast Asia. In particular, whereas the Indian CVIET haplotype has a southeast Asian origin, the SVMNT haplotype, prevalent in India, seems to have originated in Papua New Guinea and entered India through southeast Asia.     

Crystal structure and microstructure of cholesteryl oleyl carbonate

The crystal structure as well as the microstructure, i.e., size and strain, of crystallites of cholesteryl oleyl carbonate was determined from X-ray powder diffraction data. The X-ray line broadening was analyzed through the refinement of TCH-pseudo-Voigt function parameters (isotropic effects) and the refinement of multipolar functions, i.e., symmetrized cubic harmonics (anisotropic effects). The crystal structure turns out to be primitive monoclinic, space group Pc, type I monolayer having two molecules per unit cell with parameters: a = 18.921 ± 0.006 Å, b = 12.952 ± 0.003 Å, c = 9.276 ± 0.002 Å and β = 91.32 ± 0.03°. The average size of a well ground specimen of crystallites was 60 nm. The average micro-strain, e.g., 45 × 10⁻⁴ has been tentatively attributed to fatty chain conformational disorder. The unit cell parameters, including the lamellar thickness, of COC crystal is very closely similar to those of another, structurally similar cholesterol ester, e.g., cholesteryl oleate (CO) crystal, space group P2₁, type II monolayer. Type I monolayer structure has been established for COC on the basis of the intensity calculations of the XRD profiles of both CO and COC. The dipolar and structural disorder in a 4:1 molar, binary mixture of CO and COC can be accommodated in an induced smectic phase with a lamellar thickness, which is nearly equal to that of pure CO or pure COC.     

Toxicity of Crude Extracellular Products of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> on Rohu, <Emphasis Type="Italic">Labeo rohita</Emphasis> (Ham.)

In the present study the haemolytic and proteolytic activity of extracellular products (ECP) secreted from Aeromonas hydrophila (CAHH14 strain) were studied with respect to temperature and different time of incubation as well as its lethal toxicity on rohu, Labeo rohita. The strain was isolated from Catla catla (showing abdominal dropsy symptom) collected from the pond of Central Institute of Freshwater Aquaculture (CIFA), Bhubaneswar, India and was characterized on the basis of biochemical tests. The highest production of haemolysin was achieved when the bacteria was grown at 35°C for 30 h. The proteolytic activity was found to be highest when the bacterium was grown at 30°C for 36 h. The haemolytic and proteolytic toxin produced by Aeromonas hydrophila was found to be lethal to rohu (LD₅₀ 1.7 × 10⁴ cfu/ml). The lethality of ECP was decreased by heating and completely inactivated by boiling at 100°C for 10 min. This indicates that protease activity and haemolytic activity of A. hydrophila ECP was temperature dependant.