Amorphous trehalose dihydrate by cryogenic milling

Trehalose dihydrate is a safe, naturally occurring disaccharide used as a food ingredient and pharmaceutical excipient. It has been reported that room temperature milling does not lead to the formation of amorphous trehalose dihydrate. This paper reports the behaviour of trehalose dihydrate upon milling at cryogenic temperatures as studied by DSC, TGA, XRPD and Raman spectroscopy. We have demonstrated that the crystal to glass transformation for trehalose dihydrate is possible using cryogenic milling. This is the first reported example of cryogenic milling (a mild and widely applicable technique) applied to generating amorphous hydrates.     

Protein-RNA and protein-protein recognition by dual KH1/2 domains of the neuronal splicing factor Nova-1

Nova onconeural antigens are neuron-specific RNA-binding proteins implicated in paraneoplastic opsoclonus-myoclonus-ataxia (POMA) syndrome. Nova harbors three K-homology (KH) motifs implicated in alternate splicing regulation of genes involved in inhibitory synaptic transmission. We report the crystal structure of the first two KH domains (KH1/2) of?Nova-1 bound to an in?vitro selected RNA hairpin, containing a UCAG-UCAC high-affinity binding site. Sequence-specific intermolecular contacts in the complex involve KH1 and the second UCAC repeat, with the RNA scaffold buttressed by interactions between repeats. Whereas the canonical RNA-binding surface of KH2 in the above complex engages in protein-protein interactions in the crystalline state, the individual KH2 domain can sequence-specifically target the UCAC RNA element in solution. The observed antiparallel alignment of KH1 and KH2 domains in the crystal structure of the complex generates a scaffold that could facilitate target pre-mRNA looping on Nova binding, thereby potentially explaining Nova's functional role in splicing regulation.     

Cocrystal structures of diaminopimelate decarboxylase: mechanism,evolution, and inhibition of an antibiotic resistance accessory factor

Cocrystal structures of Methanococcus jannaschii diaminopimelate decarboxylase (DAPDC) bound to a substrate analog, azelaic acid, and its L-lysine product have been determined at 2.6 A and 2.0 A, respectively. This PLP-dependent enzyme is responsible for the final step of L-lysine biosynthesis in bacteria and plays a role in beta-lactam antibiotic resistance in Staphylococcus aureus. Substrate specificity derives from recognition of the L-chiral center of diaminopimelate and a system of ionic "molecular rulers" that dictate substrate length. A coupled-enzyme assay system permitted measurement of kinetic parameters for recombinant DAPDCs and inhibition constants (K(i)) for azelaic acid (89 microM) and other substrate analogs. Implications for rational design of broad-spectrum antimicrobial agents targeted against DAPDCs of drug-resistant strains of bacterial pathogens, such as Staphylococcus aureus, are discussed.     

Synthesis and SAR of novel 2-arylthiazolidinones as selective analgesic N-type calcium channel blockers

A series of new N-type (Ca(v)2.2) calcium channel blockers derived from the 'hit' structures 2-(3-bromo-4-fluorophenyl)-3-(2-pyridin-2-ylethyl)thiazolidin-4-one 9 and its 2-[4-(4-bromophenyl)pyridin-3-yl]-3-isobutyl analogue 10 is described. Extensive SAR studies using a range of synthetic approaches resulted in novel, patented compounds with IC50 values of up to 0.2 microM in an in vitro IMR32 assay, and selectivities for N/L of up to 30-fold. The new compounds described have potential in treatment of neuropathic pain.     

Continuous Evaluation of Ligand Protein Predictions: A Weekly Community Challenge for Drug Docking

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The ethics of therapeutic and reproductive human cloning.

Neither therapeutic cloning nor reproductive cloning necessarily pose insurmountable ethical obstacles. Two defences of therapeutic cloning are considered. The first defence, the argument from property, is rejected because it entails morally counter-intuitive consequences. We should prefer a 'balance of reasons' defence which leaves room for the view that human life has intrinsic value. Reproductive cloning is best defended by an appeal to the right to procreative autonomy. The sorts of harms it is feared clones will suffer are also suffered by children conceived through natural means, even when these harms were preventable. The right to reproductive autonomy disallows state control of any form of reproduction for the reasons of child welfare discussed.     

The regulation of precopulatory behavior by ovarian hormones in the female Mongolian gerbil

The hormonal regulation of precopulatory behavior in the female Mongolian gerbil was studied using two groups (N = 6) of sexually experienced females. A novel testing procedure was used which involved females living continuously with test males for several days. The test males showed either full sexual behavior (copulating males, C) or only precopulatory behavior (noncopulating males, NC). Experiment 1 investigated changes during the estrous cycle and following ovariectomy in females. Experiment 2 studied the effects of hormonal treatment of these ovariectomized females with 6 micrograms estradiol benzoate (EB) followed by 0.4 mg progesterone (P) or by 0.04 ml arachis oil. When tested with NC males, females displayed a greater range of precopulatory behavior. The patterns could be classified into three groups according to the manner of response to ovariectomy and hormone treatment. Group I patterns (approach, leave, and olfactory investigation of the male's head) were affected by neither ovariectomy nor EB treatment relative to Day 3 levels (Day 3, day preceding estrus; Day 4, estrus), but they were increased to estrous levels by EB and P. Group II patterns (darting, foot-stomping, and the present and piloerection postures) appeared only during estrus, did not appear after ovariectomy, and reappeared only after sequential EB and P treatment. Group III patterns (investigation of the male's anogenital area, allogrooming, ventral gland marking, and sand-rolling) were reduced relative to both estrus and Day 3 levels by ovariectomy and increased above Day 3 levels by EB alone; EB and P treatment further increased Group III patterns to the level of estrus. It is suggested that female precopulatory behavior patterns differ in their responsiveness to ovarian hormones. Estrogen appears to affect those patterns associated with the earliest stages of estrus (Group III).     

Antigelling and antisickling bisphenyl oligopeptides and peptide analogues have similar structural features

Single-crystal X-ray diffraction was used to determine the three-dimensional structures of two antigelling oligopeptides, L-lysyl-L-phenylalanyl-L-phenylalanine and L-phenylalanylglycylglycyl-D-phenylalanine, and two antisickling peptide analogues, L-phenylalanine benzyl ester and N-phenylacetyl-L-phenylalanine. Although these bisphenyl compounds are chemically quite different from one another, they demonstrate unusual structural similarities: The molecules have compact conformations in which the two phenyl rings are positioned approximately 5 A apart with interplanar angles approaching 90 degrees, thereby making intramolecular edge-to-face interactions. In addition, the polar atoms, nitrogen and oxygen, are in close proximity without forming intramolecular hydrogen bonds. The relative spatial distribution of polar and nonpolar atoms renders the structures compact and amphipathic. The intramolecular edge-to-face interaction between two aromatic rings, which brings a hydrogen atom with relative positive charge near the pi-electron cloud with relative negative charge, is enthalpically favorable and maintains the molecules in a compact and amphipathic conformation. Nonbonded potential energy calculations were used to characterize the energetics of the aromatic-aromatic interaction, and they showed that the observed geometry is stabilized enthalpically by a favorable interaction on the order of -1 to -2 kcal/mol. Structural differences between the two antisickling and the two antigelling agents suggest that molecular volume limits red cell membrane passage. These data provide a molecular structural framework from which to design and synthesize amphipathic bisphenyl compounds that both bind to deoxy sickle cell hemoglobin and cross the erythrocyte membrane.