Bone-targeted 2,6,9-trisubstituted purines: novel inhibitors of Src tyrosine kinase for the treatment of bone diseases

Novel bone-targeted 2,6,9-trisubstituted purine template-based inhibitors of Src tyrosine kinase are described. Drug design studies of known purine compounds revealed that both positions-2 and -6 were suitable for incorporating bone-seeking moieties. A variety of bone-targeting groups with different affinity to hydroxyapatite were utilized in the study. Compound 3d was determined to be a potent Src inhibitor and was quite selective against a panel of other protein kinases.     

Tamapin,a venom peptide from the Indian red scorpion (Mesobuthus tamulus) that targets small conductance Ca2+-activated K+ channels and afterhyperpolarization currents in central neurons

The biophysical properties of small conductance Ca(2+)-activated K(+) (SK) channels are well suited to underlie afterhyperpolarizations (AHPs) shaping the firing patterns of a conspicuous number of central and peripheral neurons. We have identified a new scorpion toxin (tamapin) that binds to SK channels with high affinity and inhibits SK channel-mediated currents in pyramidal neurons of the hippocampus as well as in cell lines expressing distinct SK channel subunits. This toxin distinguished between the SK channels underlying the apamin-sensitive I(AHP) and the Ca(2+)-activated K(+) channels mediating the slow I(AHP) (sI(AHP)) in hippocampal neurons. Compared with related scorpion toxins, tamapin displayed a unique, remarkable selectivity for SK2 versus SK1 ( approximately 1750-fold) and SK3 ( approximately 70-fold) channels and is the most potent SK2 channel blocker characterized so far (IC(50) for SK2 channels = 24 pm). Tamapin will facilitate the characterization of the subunit composition of native SK channels and help determine their involvement in electrical and biochemical signaling.     

Differential induction of mucosal and systemic antibody responses in women after nasal,rectal, or vaginal immunization: influence of the menstrual cycle

A cholera vaccine containing killed vibrios and cholera toxin B subunit (CTB) was used to compare mucosal immunization routes for induction of systemic and mucosal Ab. Four groups of women were given three monthly immunizations by the rectal immunization (R(imm)) route, nasal immunization (N(imm)) route, or vaginal immunization route during either the follicular (V-FP(imm)) or luteal (V-LP(imm)) menstrual cycle phase. N(imm) was performed with 10-fold less vaccine to determine if administration of less Ag by this route can, as in rodents, produce mucosal Ab responses comparable to those induced by higher dose R(imm) or vaginal immunization. Concentrations of Ab induced in sera and secretions were measured by ELISA. None of these routes produced durable salivary Ab responses. N(imm) induced greatest levels of CTB-specific IgG in sera. R(imm) failed to generate CTB-specific IgA in genital tract secretions. N(imm), V-FP(imm), and V-LP(imm) all produced cervical CTB-specific IgA responses comparable in magnitude and frequency. However, only V-FP(imm) induced cervical IgA2-restricted Ab to the bacterial LPS vaccine component. V-FP(imm), but not V-LP(imm), also induced CTB-specific IgA in rectal secretions. N(imm) was superior to V-FP(imm) for producing rectal CTB-specific IgA, but the greatest amounts of CTB-specific IgA and LPS-specific IgA, IgG, and IgM Ab were found in rectal secretions of R(imm) women. These data suggest that in women, N(imm) alone could induce specific Ab in serum, the genital tract, and rectum. However, induction of genital tract and rectal Ab responses of the magnitude generated by local V-FP(imm) or R(imm) will likely require administration of comparably high nasal vaccine dosages.     

Alpha-neurotoxin gene expression in Naja sputatrix: identification of a silencer element in the promoter region

Alpha-neurotoxin (alpha-NTX) from the venom of cobra, Naja sputatrix, is a highly lethal post-synaptic toxin that is responsible for the lethality caused by the venom. However, this toxin is found at low levels (3%) in the crude venom. The expression of its gene is determined by a promoter which is 90% similar to the promoter of another three-fingered toxin, cardiotoxin (CTX), which is produced in large amounts (60%) in the same venom. Functional analysis of the NTX-2 gene promoter demonstrated the presence of a silencer element of 24 nucleotides (nt -678 to -655) at its 5(') flanking region. This element has been found to play a major role in the down-regulation of NTX-2 gene expression. A point mutation on this silencer appears to attenuate its repressive property in CTX-2 gene.     

A synthetic weak neurotoxin binds with low affinity to Torpedo and chicken alpha7 nicotinic acetylcholine receptors.

Weak neurotoxins from snake venom are small proteins with five disulfide bonds, which have been shown to be poor binders of nicotinic acetylcholine receptors. We report on the cloning and sequencing of four cDNAs encoding weak neurotoxins from Naja sputatrix venom glands. The protein encoded by one of them, Wntx-5, has been synthesized by solid-phase synthesis and characterized. The physicochemical properties of the synthetic toxin (sWntx-5) agree with those anticipated for the natural toxin. We show that this toxin interacts with relatively low affinity (K(d) = 180 nm) with the muscular-type acetylcholine receptor of the electric organ of T. marmorata, and with an even weaker affinity (90 microm) with the neuronal alpha7 receptor of chicken. Electrophysiological recordings using isolated mouse hemidiaphragm and frog cutaneous pectoris nerve-muscle preparations revealed no blocking activity of sWntx-5 at microm concentrations. Our data confirm previous observations that natural weak neurotoxins from cobras have poor affinity for nicotinic acetylcholine receptors.     

Trait variation in juvenile plants from the soil seed bank of temperate forests in relation to macro- and microclimate

Aim

The soil seed bank is a key component of the biodiversity of plant communities, but various aspects of its functioning in temperate forest ecosystems are still unknown. We here adopted a trait-based approach to investigate the effects of macro- and microclimatic gradients on the juvenile plant communities from the realized seed bank of two types of European temperate forest.

Location

Oak-dominated forests in Italy and Belgium.

Methods

We analysed the variation of key functional traits (plant height, leaf area, leaf dry weight, specific leaf area and leaf number) of juvenile plants from the realised soil seed bank in relation to elevation (from 0 to 800 m a.s.l.), forest type (thinned and unthinned forest) and distance to the forest edge. We translocated soil samples from the forest core to the edge (and vice versa) and from high- to low-elevation forests to test the effects of edge and warming respectively.

Results

Taller communities developed at the forest edge due to higher light availability and warmer temperatures. The translocation from the core to the edge did not significantly modify mean trait values. Instead, the shadier and cooler microclimate of the forest core reduced the mean leaf area, mean dry weight, height and leaf number in the communities realised from the edge soil. The translocation from high- to lowland forests led to increased values for all traits (except specific leaf area). Edge vs core trait variation was more driven by intraspecific variability, whereas the translocation from high- to low-elevation forests caused trait changes mostly due to species turnover.

Conclusions

Global warming might result in a functional shift of the understorey due to both an early filtering effect on the seedlings from soil seed banks and their adaptive trait adjustments to temperature increase. Furthermore, our study underpins the importance of edge vs core microclimate in driving the functional composition of the realised soil seed bank.     

Plasmodium falciparum hypoxanthine guanine phosphoribosyltransferase. Stability studies on the product-activated enzyme

Hypoxanthine guanine phosphoribosyltransferases (HGPRTs) catalyze the conversion of 6-oxopurine bases to their respective nucleotides, the phosphoribosyl group being derived from phosphoribosyl pyrophosphate. Recombinant Plasmodium falciparum HGPRT, on purification, has negligible activity, and previous reports have shown that high activities can be achieved upon incubation of recombinant enzyme with the substrates hypoxanthine and phosphoribosyl pyrophosphate [Keough DT, Ng AL, Winzor DJ, Emmerson BT & de Jersey J (1999) Mol Biochem Parasitol98, 29-41; Sujay Subbayya IN & Balaram H (2000) Biochem Biophys Res Commun279, 433-437]. In this report, we show that activation is effected by the product, Inosine monophosphate (IMP), and not by the substrates. Studies carried out on Plasmodium falciparum HGPRT and on a temperature-sensitive mutant, L44F, show that the enzymes are destabilized in the presence of the substrates and the product, IMP. These stability studies suggest that the active, product-bound form of the enzyme is less stable than the ligand-free, unactivated enzyme. Equilibrium isothermal-unfolding studies indicate that the active form is destabilized by 2-3 kcal x mol(-1) compared with the unactivated state. This presents a unique example of an enzyme that attains its active conformation of lower stability by product binding. This property of ligand-mediated activation is not seen with recombinant human HGPRT, which is highly active in the unliganded state. The reversibility between highly active and weakly active states suggests a novel mechanism for the regulation of enzyme activity in P. falciparum.     

Structure and phylogeny of the venom group I phospholipase A(2) gene

Phospholipases A(2) (PLA(2)s) catalyzing the hydrolysis of phospholipids form a family of proteins with diverse physiological and pharmacological properties. While there have been several reports on the cloning of PLA(2) cDNAs, very few studies have been carried out on the PLA(2) genes and, most importantly, no information has been available on the gene structure and function of group I venom PLA(2). This study, on the PLA(2) gene from a spitting cobra, besides being the very first report on any venom group I PLA(2) gene, constitutes the missing link in the biology and evolution of phospholipases. The 4-kb gene consists of four exons and three introns and resembles the human pancreatic PLA(2) gene. However, the size of intron 3 in particular is much smaller than that in the pancreatic gene. Interestingly, the information for the toxic and most of the pharmacological properties of the venom PLA(2) can be attributed to the end of exon 3 and the whole of exon 4 of the gene. This functional delineation fits in well with the theory of adaptive evolution exhibited by the venom PLA(2)s. We also show that the mammalian pancreatic and elapid PLA(2)s have similar paths of evolution (probably following gene duplication) from a common ancestral gene. Venom group II phospholipases, although evolved from the same ancestor, diverged early in evolution from the group I PLA(2) genes. Intriguingly, CAT reporter gene assays and DNase 1 footprinting studies on the promoter and its deletion constructs using CHO and HepG2 cell lines identified the possible involvement of cis elements such as Sp1, AP2, gamma-IRE, and (TG)(12) repeats in the expression of the gene in a tissue-specific manner.     

Synthesis, structure and DNA interaction of cobalt(III) bis-complexes of 1,3-bis(2-pyridylimino)isoindoline and 1,4,7-triazacyclononane

The complex [CoL(2)](ClO(4)).MeOH (1), where HL is the tridentate 3N ligand 1,3-bis(2-pyridylimino)isoindoline, has been isolated and its X-ray crystal structure successfully determined. It possesses a distorted octahedral structure in which both the ligands are coordinated meridionally to cobalt(III) via one deprotonated isoindoline (L(-)) and two pyridine nitrogen atoms. Interestingly, the average dihedral angle between pyridine and isoindoline rings is 25.9 degrees , indicating that the ligand is twisted upon coordination to cobalt(III). The interaction of the complex with calf-thymus DNA has been studied using various spectral methods and viscosity and electrochemical measurements. For comparison, the DNA interaction of [Co(tacn)(2)]Cl(3) (2), where tacn is facially coordinating 1,4,7-triazacyclononane, has been also studied. The ligand-based electronic spectral band of 1 and the N(sigma)-->Co(III) charge transfer band of 2 exhibit moderate hypochromism with small or no blue shift on interaction with DNA. The intrinsic binding constants calculated reveal that the monopositive complex ion [CoL(2)](+) exhibits a DNA-binding affinity lower than the tripositive complex ion [Co(tacn)(2)](3+). The steric clashes with DNA exterior caused by the second L(-) ligand bound to cobalt(III), apart from the lower overall positive charge on the [CoL(2)](+) complex, dictates its DNA-binding mode to be surface binding rather than partial intercalative interaction expected of the extended aromatic chromophore of deprotonated isoindoline anion. An enhancement in relative viscosity of CT DNA on binding to 1 is consistent with its DNA surface binding. On the other hand, a slight decrease in viscosity of CT DNA was observed on binding to 2 revealing that the smaller cation leads to bending (kinking) and hence shortening of DNA chain length. The electrochemical studies indicate that the DNA-bound complexes are stabilised in the higher Co(III) rather than the lower Co(II) oxidation state, suggesting the importance of electrostatic forces of DNA interaction.     

Epitope mapping from real time kinetic studies — Role of cross-linked disulphides and incidental interacting regions in affinity measurements: Study with human chorionic gonadotropin and monoclonal antibodies

Real time kinetic studies were used to map conformational epitopes in human chorionic gonadotropin (hCG) for two monoclonal antibodies (MAbs). The epitopes were identified in the regions (α5-14 and α55-62). The association rate constant (k₊₁) was found to be altered by chemical modification of hCG, and the ionic strength of the reaction medium. Based on these changes, we propose the presence of additional interactions away from the epitope-paratope region in the hCG-MAb reaction. We have identified such incidental interacting regions (IIRs) in hCG to be the loop region α35-47 and α60-84. The IIRs contribute significantly towards theK _A of the interaction. Therefore, in a macromolecular interaction of hCG and its MAb,K _A is determined not only by epitopeparatope interaction but also by the interaction of the nonepitopic-nonparatopic IIRs. However, the specificity of the interaction resides exclusively with the epitope-paratope pair.