Effect of UV Stress on the Structure and Function of Pro-apoptotic Bid and Anti-apoptotic Bcl-xl proteins.

Ultraviolet C (UV-C) radiation induces apoptosis in mammalian cells via the mitochondrion-mediated pathway. The Bcl-2 family of proteins are the regulators of the mitochondrial pathway of apoptosis and appears responsive to UV-C radiation. It is unknown how the structure and, effectively, the function of these proteins are directly impacted by UV-C exposure. Here, we present the effect of UV-C irradiation on the structure and function of pro-apoptotic Bid-FL and anti-apoptotic Bcl-xlΔC proteins. Using a variety of biophysical tools, we show that, following UV-C irradiation, the structures of Bcl-xlΔC and Bid-FL are irreversibly altered. Bcl-xLΔC is found to be more sensitive to UV stress than Bid-FL Interestingly, UV-C exposure shows dramatic chemical shift perturbations in consequence of dramatic structural perturbations (α-helix to ß-sheet) in the BH3- binding region, a crucial segment of Bcl-xlΔC. Furter it has been shown that UV-exposed Bcl-xlΔC has reduced efficacy of its interactions with pro-apoptotic tBid.

Curcumin, a potential initiator of apoptosis via direct interactions with Bcl-xL and Bid.

Apoptosis is a naturally occurring process during the growth and development of multicellular organisms and is increasingly active during times of cellular stress such as in response to intracellular DNA damage when removal of the host cell is paramount to prevent cancer. Unfortunately, once formed, cancer cells become impervious to apoptosis, creating a desperate need to identify an approach to induce apoptosis in these cells. An attractive option is to focus efforts on developing and locating compounds which activate apoptosis using natural compounds. Curcumin is a natural component in turmeric and is well-known for its pharmacological effects in preventing and combating many ailments and has been shown to decrease the rapid proliferation of a wide variety of tumor cells. However, to date, the apoptotic intermediates and interactions through which curcumin exerts its cytotoxic effects are unknown. Motivated by reports linking the intracellular modulation of the concentrations of Bid and Bcl-xL, following curcumin administration to cancer cells, we set out to probe for potential intermolecular interactions of these proteins with curcumin. Using several biophysical techniques, most notably, fluorescence, circular dichroism and nuclear magnetic resonance spectroscopy, we reveal binding interactions of curcumin with both Bcl-xLΔC and full-length Bid (Bid-FL) and prove that this binding is hydrophobically driven and localized to well-known functional regions of each protein. Specifically, our NMR studies show that while Bid-FL interacts with curcumin through its hydrophobic and pore forming helices (α6-α7), Bcl-xLΔC interacts with curcumin via its BH3 binding pocket (α2-α3-α4-α5), a critical region for mediating apoptosis.

Simultaneous measurement of 1HC/N-R2's for rapid acquisition of backbone and sidechain paramagnetic relaxation enhancements (PREs) in proteins.

Paramagnetic relaxation enhancements (PREs) are routinely used to provide long-range distance restraints for the determination of protein structures, to resolve protein dynamics, ligand-protein binding sites, and lowly populated species, using Nuclear Magnetic Resonance Spectroscopy (NMR). Here, we propose a simultaneous 1H-15 N, 1H-13C SESAME based pulse scheme for the rapid acquisition of 1HC/N-R2 relaxation rates for the determination of backbone and sidechain PREs of proteins. The 1HN-R2 rates from the traditional and our approach on Ubiquitin (UBQ) are well correlated (R2 = 0.99), revealing their potential to be used quantitatively. Comparison of the S57C UBQ calculated and experimental PREs provided backbone and side chain Q factors of 0.23 and 0.24, respectively, well-fitted to the UBQ NMR structure, showing that our approach can be used to acquire accurate PRE rates from the functionally important sites of proteins but in at least half the time as traditional methods.

Solvent saturation transfer to proteins (SSTP) for structural and functional characterization of proteins.

Protein structure determination using NMR is dependent on experimentally acquired distance restraints. Often, however, an insufficient number of these restraints are available for determining a protein's correct fold, much less its detailed three-dimensional structure. In consideration of this problem, we propose a simple means to acquire supplemental structural restraints from protein surface accessibilities using solvent saturation transfer to proteins (SSTP), based on the principles of paramagnetic chemical-exchange saturation transfer. Here, we demonstrate the utility of SSTP in structure calculations of two proteins, TSG101 and ubiquitin. The observed SSTP was found to be directly proportional to solvent accessibility. Since SSTP does not involve the direct excitation of water, which compromises the analysis of protein protons entangled in the breadth of the water resonance, it has an advantage over conventional water-based magnetization transfers. Inclusion of structural restraints derived from SSTP improved both the precision and accuracy of the final protein structures in comparison to those determined by traditional approaches, when using minimal amounts of additional structural data. Furthermore, we show that SSTP can detect weak protein-protein interactions which are unobservable by chemical shift perturbations.

Curcumin and kaempferol prevent lysozyme fibril formation by modulating aggregation kinetic parameters.

Interaction of small molecule inhibitors with protein aggregates has been studied extensively, but how these inhibitors modulate aggregation kinetic parameters is little understood. In this work, we investigated the ability of two potential aggregation inhibiting drugs, curcumin and kaempferol, to control the kinetic parameters of aggregation reaction. Using thioflavin T fluorescence and static light scattering, the kinetic parameters such as amplitude, elongation rate constant and lag time of guanidine hydrochloride-induced aggregation reactions of hen egg white lysozyme were studied. We observed a contrasting effect of inhibitors on the kinetic parameters when aggregation reactions were measured by these two probes. The interactions of these inhibitors with hen egg white lysozyme were investigated using fluorescence quench titration method and molecular dynamics simulations coupled with binding free energy calculations. We conclude that both the inhibitors prolong nucleation of amyloid aggregation through binding to region of the protein which is known to form the core of the protein fibril, but once the nucleus is formed the rate of elongation is not affected by the inhibitors. This work would provide insight into the mechanism of aggregation inhibition by these potential drug molecules.

Molecular study of binding of Plasmodium ribosomal protein P2 to erythrocytes.

The ribosomal protein P2 of Plasmodium falciparum, (PfP2), performs certain unique extra-ribosomal functions. During the few hours of cell-division, PfP2 protein moves to the external surface of the infected erythrocytes (IE) as an SDS-resistant oligomer, and at that stage treatment with specific anti- PfP2 antibodies results in an arrest of the parasite cell-division. Amongst the oligomeric forms of PfP2, mainly the homo-tetramer is peripherally anchored on the external surface of the IE. To study the anchoring of PfP2 tetramer on IE-surface, we have explored the binding properties of PfP2 protein. Using NMR and erythrocyte pull-down studies, here we report that the homo-tetrameric PfP2 protein interacted specifically with erythrocytes and not leukocytes. The hydrophobic N-terminal 72 amino acid region is the major interacting domain. The binding of P2 to RBCs was neuraminidase resistant, but trypsin sensitive. The RBC binding was exclusive to the Plasmodium PfP2 protein as even the homologous protein of the closely related Apicomplexan parasite Toxoplasma gondii TgP2 protein did not interact with erythrocytes. Pull down assays, immunoprecipitation and mass spectrometry data showed that erythrocytic Band 3 protein is a possible interactor of Plasmodium PfP2 protein on the erythrocyte surface.

Intranasal cabergoline: pharmacokinetic and pharmacodynamic studies.

Aims of this investigation were to prepare and characterize cabergoline intranasal microemulsion formulations, determine brain drug delivery through biodistribution using technetium-99m (99mTc) as a tracer, and assess its performance pharmacodynamically in weight control. Cabergoline microemulsions of different compositions were prepared by water titration method and characterized for globule size and zeta potential. Microemulsion with maximum drug solubilization and stability was considered optimal and taken for further studies with or without addition of mucoadhesive agent. Pharmacokinetics of optimized 99mTc-labeled cabergoline formulations and 99mTc-labeled drug solution were studied by estimating radioactivity in brain and blood of albino rats post intranasal, intravenous, and oral administrations. To confirm localization of drug in brain following intranasal, intravenous, and oral administrations, gamma scintigraphy imaging was also performed. To assess weight control performance of formulations, body weight, white adipose tissue mass, serum lipids, leptin, and prolactin were determined before and after 40 days of intranasal administrations of these formulations to Wistar rats. Microemulsions were found to be stable both physically and chemically when stored at various stress conditions. Brain/blood uptake ratios, drug targeting efficiency, and direct drug transport were found to be highest for drug mucoadhesive microemulsion followed by drug microemulsion and drug solution post-intranasal administration compared to intravenous drug microemulsion. Significant (p<0.05) reduction in assessed pharmacodynamic parameters was observed after intranasal administration of mucoadhesive microemulsion against control group. The results of the studies conclusively demonstrate that intranasal microemulsion formulations developed in this investigation are stable and can deliver cabergoline selectively and in higher amounts to the brain compared to both drug administrations as a solution intranasally or microemulsion intravenously. The results also demonstrate reduction in weight, adipose tissue mass, serum lipids, and serum prolactin after intranasal administration of drug microemulsion. Hence, long-term studies in at least two more animal models followed by extensive clinical evaluation can safely result into a product for clinical use.

Intranasal mucoadhesive microemulsion of tacrine to improve brain targeting.

The aim of the investigation was to prepare and characterize microemulsion/mucoadhesive microemulsion of tacrine (TME/TMME), assess its pharmacokinetic and pharmacodynamic performances for brain targeting and for improvement in memory in scopolamine-induced amnesic mice. The TME was prepared by the titration method and characterized. Biodistribution of tacrine solution and formulations after intravenous and intranasal administrations were evaluated using 99m Tc as marker. From the data, the pharmacokinetic parameters, drug targeting efficiency, and direct nose-to-brain drug transport were calculated. To confirm drug localization in brain gamma scintigraphy in rabbits was performed. Lower Tmax values (60 min) after intranasal compared with intravenous administration (120 min) suggested selective nose-to-brain transport. The brain bioavailability of tacrine after intranasal TMME compared with intranasal tacrine solution was found to be 2-fold higher indicating larger extent of distribution of the drug to brain with intranasal TMME. Rabbit brain scintigraphy also showed higher uptake of drug into the brain after intranasal administration. The results demonstrated rapid and larger extent of transport of tacrine into the mice brain and fastest regain of memory loss in scopolamine-induced amnesic mice after intranasal TMME. Hence, results are suggestive of possible role of intranasal tacrine delivery in treating Alzheimer's patients.

Synthesis, characterization and biological activity of Schiff base analogues of indole-3-carboxaldehyde.

Eight novel heterocyclic Schiff bases derived from the condensation reactions of indole 3-carboxaldehyde with different l-amino acids (histidine, glutamic acid, aspartic acid, leucine, valine) as well as with some aminophenols, have been synthesized and characterized by various spectroscopic methods (IR, MS, (1)H NMR). Schiff base derivatives of indole 3-carboxaldehyde were labeled with (99m)Tc and radiochemical purity was above 97% which is ascertained by instant thin layer chromatography using different solvent conditions. Stability studies of all the derivatives of indole 3-carboxaldehyde was determined under physiological conditions and were stable for more than 24h. Blood clearance showed a quick wash out from the circulation and biological half life was found to be t((1/2))(F)=1h 15min; t((1/2))(S)=10h 05min. Excellent quality radioimages of tumor bearing mice were recorded showing rapid clearance of background activity, visualization of tumor at 3h and clearance from kidneys of histidine analogue which was further evidenced in biodistribution studies. Antimicrobial activity of these Schiff base compounds was evaluated against Bacillus subtilis, Pseudomonas fluorescence, Staphylococcus aureus, Aspergillus niger, Candida albicans and Trichophyton rubrum.

Radiolabeling, pharmacoscintigraphic evaluation and antiretroviral efficacy of stavudine loaded 99mTc labeled galactosylated liposomes.

The study was aimed to optimize radiolabeling with 99mTc, to determine the antiretroviral activity and to study the biodistribution of 99mTc labeled galactosylated liposomes loaded with stavudine. Liposomes were prepared using reverse-phase evaporation method followed by extrusion through 200nm polycarbonate membranes. The galactosylated liposomes were assessed for in vitro ligand-specific activity and the aggregation of galactosylated liposomes was found to increase as lectin concentration was increased from 5microg/ml to 30microg/ml. Free stavudine and stavudine loaded plain and galactosylated liposomes were radiolabeled with 99mTc by direct labeling method using stannous chloride as a reducing agent. Labeling method was optimized for stannous chloride quantity to achieve maximum labeling efficiency >95%. Antiretroviral activity was determined using human immunodeficiency virus-1 (HIV) infected MT2 cell line. A dose-dependent inhibition of p24 production was observed upon treatment of HIV-1 infected MT2 cells with stavudine loaded liposomes and galactosylated liposomes. Scintigraphic imaging and quantitative biodistribution of 99mTc labeled drug and liposomes showed that liposomal formulations were better taken up by the liver and spleen. Free drug solution was cleared from the blood. Further, a significantly higher (P<0.05) liver and spleen retention was observed over a period of 24h in case of galactosylated liposomes as compared to free drug and plain liposomes. Reduced uptake of the galactosylated liposomes in bone and higher and prolonged accumulation in mononuclear phagocyte system (MPS)-rich organs indicates the excellent potential of this formulation in the treatment of HIV infection.

Modulation of ganciclovir intestinal absorption in presence of absorption enhancers.

The purpose of this investigation was to study the influences of absorption enhancers in increasing oral bioavailability of Ganciclovir (GAN) by assessing the transepithelial permeation across cell monolayers in vitro and bioavailability in rats in vivo. The permeation of GAN across Caco-2 and MDCK cell monolayers in the absence/presence of dimethyl-beta-cyclodextrin (DMbetaCD), chitosan hydrochloride (CH), sodium lauryl sulphate (SLS), and their combinations was studied for a 2-h period. GAN was administered to rats in absence/presence of absorption enhancers and drug contents in plasma were estimated. We found that the apparent permeability coefficient (Papp) of GAN in absence of absorption enhancers (control) were 0.261 +/- 0.072 x 10(-6) and 0.486 +/- 0.063 x 10(-6) cm/s in Caco-2 and MDCK cell monolayers, respectively, whereas in the presence of DMbetaCD, CH, SLS, and their combinations, Papp of GAN increased by 5- to 25-fold and 7- to 33-fold as compared to control in Caco-2 and MDCK cell monolayers, respectively. However, in rats, the maximum enhancement in bioavailability of GAN during coadministration of these absorption enhancers was only fivefold compared to GAN control. To conclude, the absorption enhancers-DMbetaCD, CH, SLS, and their combinations demonstrated significant improvement in transepithelial permeation and bioavailability of GAN.

Role of 99mTc-mannitol and 99mTc-PEG in the assessment of paracellular integrity of cell monolayers.

AIM: To assess the role of 99mTc-mannitol and 99mTc-polyethylene glycol 4000 in the evaluation of paracellular integrity of Caco-2 and Madine-Darby canine kidney (MDCK) cell monolayers, and confirm it in the presence of absorption promoters. METHODS: Radiolabelling of mannitol and polyethylene glycol was performed by a simple reduction method. Transepithelial electrical resistance values were measured to gain information regarding the integrity of tight junctions of Caco-2 and MDCK cell monolayers. Permeabilities of 99mTc-mannitol/99mTc-polyethylene glycol across cell monolayers were studied in the absence and presence of absorption promoters, namely dimethyl-beta-cyclodextrin, chitosan hydrochloride and sodium lauryl sulfate, and during recovery studies to assess paracellular integrity. RESULTS: Values for the apparent permeability coefficient (Papp) of Tc-mannitol were found to be 0.286 x 10 cm x s(-1) and 0.507 x 10 cm x s(-1) in Caco-2 and MDCK cell monolayers, respectively, whereas corresponding values for 99mTc-polyethylene glycol were 0.046 x 10 cm x s(-1) and 0.065 x 10 cm x s(-1). The insignificant Papp values of the marker molecules demonstrated the paracellular integrity of the cell monolayers. Significant increases in the Papp values in the presence of absorption promoters and their combinations due to opening of paracellular pathways and a return of Papp values to almost baseline values during recovery studies confirm the role of these marker molecules in the assessment of paracellular integrity of cell monolayers. CONCLUSION: 99mTc-labelled marker molecules can be attractive, useful and viable alternatives to the conventionally used markers in the assessment of paracellular integrity because of the absence of tissue-damaging corpuscular radiation and the ease of production of radiochemically pure and stable molecules at a reasonable cost.

Nose-to-brain delivery of tacrine.

In the treatment of Alzheimer's disease tacrine, a cholinesterase inhibitor, is not the drug of choice due to its low oral bioavailability, extensive hepatic first-pass effect, rapid clearance from the systemic circulation, pronounced hepatotoxicity, and the availability of drugs better than tacrine in the same pharmacological class. Hence, the aim of this investigation was to ascertain the possibility of direct nose-to-brain delivery of tacrine to improve bioavailability, to avoid the first-pass effect and to minimize hepatotoxicity. Tacrine solution (TS) in propylene glycol was radiolabelled with (99m)Tc (technetium) and administered in BALB/c mice intranasally (i.n.) and intravenously (i.v.). Drug concentrations in blood and brain were determined at predetermined time intervals post dosing. Drug targeting efficiency (DTE %) and the brain drug direct transport percentage (DTP %) were calculated to evaluate the brain targeting efficiency. Brain scintigraphy imaging in rabbits was performed to ascertain the uptake of the drug into the brain. Tacrine solution was effectively labelled with (99m)Tc and was found to be stable and suitable for in-vivo studies. Following intranasal administration tacrine was delivered quickly (T(max) 60 min) to the brain compared with intravenous administration (T(max) 120 min). The brain/blood ratios of the drug were found to be higher for [(99m)Tc]TS(i.n.) compared with [(99m)Tc]TS(i.v.) at all time points. The DTE (207.23%) and DTP (51.75%) following intranasal administration suggested that part of tacrine was directly transported to brain from the nasal cavity. Rabbit brain scintigraphy imaging showed higher uptake of the drug into the brain following intranasal administration compared with intravenous administration. The results showed that tacrine could be directly transported into the brain from the nasal cavity and intranasal administration resulted in higher bioavailability of drug with reduced distribution into non-targeted tissues. This selective localization of tacrine in the brain may be helpful in reducing dose, frequency of dosing and dose-dependent side effects, and may prove an interesting new approach in delivery of the drug to the brain for the treatment of Alzheimer's disease.

Development and characterization of hyaluronic acid-anchored PLGA nanoparticulate carriers of doxorubicin.

A novel hyaluronic acid-poly(ethylene glycol)-poly(lactide-co-glycolide) (HA-PEG-PLGA) copolymer was synthesized and characterized by infrared and nuclear magnetic resonance spectroscopy. The nanoparticles of doxorubicin (DOX)-loaded HA-PEG-PLGA were prepared and compared with monomethoxy(polyethylene glycol) (MPEG)-PLGA nanoparticles. Nanoparticles were prepared using drug-to-polymer ratios of 1:1 to 1:3. Drug-to-polymer ratio of 1:1 is considered the optimum formulation on the basis of low particle size and high entrapment efficiency. The optimized nanoparticles were characterized for morphology, particle size measurements, differential scanning calorimetry, x-ray diffractometer measurement, drug content, hemolytic toxicity, subacute toxicity, and in vitro DOX release. The in vitro DOX release study was performed at pH 7.4 using a dialysis membrane. HA-PEG-PLGA nanoparticles were able to sustain the release for up to 15 days. The tissue distribution studies were performed with DOX-loaded HA-PEG-PLGA and MPEG-PLGA nanoparticles after intravenous (IV) injection in Ehrlich ascites tumor-bearing mice. The tissue distribution studies showed a higher concentration of DOX in the tumor as compared with MPEG-PLGA nanoparticles. The in vivo tumor inhibition study was also performed after IV injection of DOX-loaded HA-PEG-PLGA nanoparticles up to 15 days. DOX-loaded HA-PEG-PLGA nanoparticles were able to deliver a higher amount of DOX as compared with MPEG-PLGA nanoparticles. The DOX-loaded HA-PEG-PLGA nanoparticles reduced tumor volume significantly as compared with MPEG-PLGA nanoparticles.

Sustained ocular drug delivery from a temperature and pH triggered novel in situ gel system.

Various ocular diseases like glaucoma, conjunctivitis, and dry eye syndrome require frequent drug administration. Poor ocular bioavailability of drugs (< 1%) from conventional eye drops is due mainly to the precorneal loss factors that include rapid tear turnover, nonproductive absorption, transient residence time in the cul-de-sac, and the relative impermeability of the drugs to corneal epithelial membrane. These problems may be overcome by the use of in situ gel-forming systems that are instilled as drops into the eye and undergo a sol-gel transition in the cul-de-sac. Our present work describes the formulation and evaluation of an ocular delivery system of timolol maleate based on the concept of both temperature and pH-triggered in situ gelation. Pluronic F-127 (a thermosensitive polymer) in combination with chitosan (pH-sensitive polymer also acts as permeation enhancer) was used as gelling agent. The developed formulation was characterized for various in vitro parameters e.g., clarity, gelation temperature and pH, isotonicity, sterility, rheological behavior, drug release profile, transcorneal permeation profile, and ocular irritation. Developed formulation was clear, isotonic solution, that converted into gel at temperatures above 35 degrees C and pH 6.9-7.0. A significant higher drug transport across corneal membrane and increased ocular retention time was observed using the developed formulation. The developed system is a viable alternative to conventional eye drops for the treatment of glaucoma and various other ocular diseases.

Conformational Heterogeneity in the Activation Mechanism of Bax.

Bax is known for its pro-apoptotic role within the mitochondrial pathway of apoptosis. However, the mechanism for transitioning Bax from cytosolic to membrane-bound oligomer remains elusive. Previous nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) studies defined monomeric Bax as conformationally homogeneous. Yet it has recently been proposed that monomeric Bax exists in equilibrium with a minor state that is distinctly different from its NMR structure. Here, we revisited the structural analysis of Bax using methods uniquely suited for unveiling "invisible" states of proteins, namely, NMR paramagnetic relaxation enhancements and EPR double electron-electron resonance (DEER). Additionally we examined the effect of glycerol, the co-solvent of choice in DEER studies, on the structure of Bax using NMR chemical-shift perturbations and residual dipolar couplings. Based on our combined NMR and EPR results, Bax is a conformationally homogeneous protein prior to its activation.

Synthesis of trans-1,2-cyclohexyldinitrilo tetramethylene phosphonic acid and its radiolabelling with 99mTc for the detection of skeletal metastases.

BACKGROUND AND AIM: The development of bone-seeking radiopharmaceuticals for the detection of malignant bone lesions could further improve the diagnostic accuracy of routine bone scanning. This study aimed to provide a convenient synthesis of trans-1,2-cyclohexylenedinitrilo tetramethylene phosphonic acid (CDTMP) and an improved preparation of its (99m)Tc complex. METHODS: CDTMP was prepared from trans-1,2-cyclohexyldinitrilotetraacetic acid by reaction with phosphorus trichloride and it was labelled with (99m)Tc. Toxicity and biodistribution studies were carried out in BALB/c mice, while blood clearance and bone scintigraphy studies were carried out in rabbits. (99m)Tc-CDTMP was evaluated for the detection of malignant bone lesions in 11 patients. Bone scintigraphy (a methylene diphosphonate scan) was performed to detect metastases at diagnosis and follow-up. RESULTS: The radiolabelling efficiency was found to be >97% and the stability in serum indicated that (99m)Tc remained bound to the chelate, CDTMP, for up to 24 h. Blood clearance showed a quick wash-out from the circulation and the biological half-lives (t12) were 55 min (F) and 8 h 48 min (S). The LD50 was 110 mg.kg(-1) as determined by toxicity studies. The drug was excreted mainly through renal route and the accumulation of (99m)Tc-CDTMP in bone was 7.69+/-0.65%ID/g at 1 h. The mean ratio of bone lesion to soft tissue was 6.8+/-0.69 and of bone lesion to normal bone was 5.67+/-0.82. Visual image analysis of (99m)Tc-CDTMP was clinically comparable to the interpretation of imaging studies with (99m)Tc-MDP. CONCLUSION: These preliminary data support increased bone uptake by the tetraphosphonate complex of (99m)Tc. This suggests that CDTMP complexed with therapeutic radionuclides should be evaluated for therapy of skeletal metastases.

EWGWS insert in Plasmodium falciparum ookinete surface enolase is involved in binding of PWWP containing peptides: Implications to mosquito midgut invasion by the parasite.

There are multiple stages in the life cycle of Plasmodium that invade host cells. Molecular machinery involved is such host-pathogen interactions constitute excellent drug targets and/or vaccine candidates. A screen using a phage display library has previously demonstrated presence of enolase on the surface of the Plasmodium ookinete. Phage-displayed peptides that bound to the ookinete contained a conserved motif (PWWP) in their sequence. Here, direct binding of these peptides with recombinant Plasmodium falciparum enolase (rPfeno) was investigated. These peptides showed specific binding to rPfeno, but failed to bind to other enolases. Plasmodium spp enolases are distinct in having an insert of five amino acids ((104)EWGWS(108)) that is not found in host enolases. The possibility of this insert being the recognition motif for the PWWP containing peptides was examined, (i) by comparing the binding of the peptides with rPfeno and a deletion variant Δ-rPfeno lacking (104)EWGWS(108), (ii) by measuring the changes in proton chemical shifts of PWWP peptides on binding to different enolases and (iii) by inter-molecular docking experiment to locate the peptide binding site. Results from these studies showed that the pentapeptide insert of Pfeno indeed constitutes the binding site for the PWWP domain containing peptide ligands. Search for sequences homologous to phage displayed peptides among peritrophic matrix proteins resulted in identification of perlecan, laminin, peritrophin and spacran. The possibility of these PWWP domain-containing proteins in the peritrophic matrix of insect gut to interact with ookinete cell surface enolase and facilitate the invasion of mosquito midgut epithelium is discussed.

Radiolabeling and biological evaluation of DOTA-Ph-Al derivative conjugated to anti-EGFR antibody ior egf/r3 for targeted tumor imaging and therapy.

An appropriate bifunctional chelating agent namely DOTA-Ph-Al was developed for the conjugation with biological vectors (anti EGFr antibody). We hereby report the synthesis of p-bromoacetamidobenzyl derivative of DOTA and its conjugation to monoclonal antibody anti-EGFR ior egf/r3. Immunoconjugate was prepared by conjugation of p-bromoacetamidobenzyl derivative of DOTA with ior egf/r3. Modified antibody was purified by size exclusion chromatography. DOTA-Ph-Al-ior egf/r3 exhibited quantitative 99mTc-labeling (>96%) with specific activity 10-20 mCi/mg of protein and 90Y-labeling with specific activity 2-5 mCi/mg. Immunoreactivity was determined by flow cytometry. Receptor ligand assay on murine cell line EAT and human tumor cell line U-87MG showed Kd = 2.87 nM and 4.86 nM respectively. The stability in serum indicated that 99mTc remained bound to antibodies up to 24h and 98% 90Y was associated with the mAb for five days. Biodistribution characteristics of Ab-conjugate radiolabeled to 99mTc and 90Y radionuclide was examined in BALB/c mice grafted with EAT and athymic mice with U-87MG cell line demonstrated high tumor uptake with 5.5 +/- 1.3 and 7.85 +/- 1.2%ID/g at four and 24 h for 99mTc- DOTA-Ph-AI-ior egf/r3 in EAT tumors after post injection respectively. Maximal radiotracer uptake peaked 17.6 +/- 2.5%ID/g in EAT tumor and 12.89 +/- 0.66% ID/g in U-87MG tumor at 48h for 90Y. The drug excreted through renal routes as the activity in the kidneys was 13.42 +/- 0.33%ID/g at 1 h and 4.51 +/- 1.2%ID/g at 4 h for 99mTc- DOTA-Ph-Al-ior egf/r3.