Potentiation of M1 Muscarinic Receptor Reverses Plasticity Deficits and Negative and Cognitive Symptoms in a Schizophrenia Mouse Model.

Schizophrenia patients exhibit deficits in signaling of the M1 subtype of muscarinic acetylcholine receptor (mAChR) in the prefrontal cortex (PFC) and also display impaired cortical long-term depression (LTD). We report that selective activation of the M1 mAChR subtype induces LTD in PFC and that this response is completely lost after repeated administration of phencyclidine (PCP), a mouse model of schizophrenia. Furthermore, discovery of a novel, systemically active M1 positive allosteric modulator (PAM), VU0453595, allowed us to evaluate the impact of selective potentiation of M1 on induction of LTD and behavioral deficits in PCP-treated mice. Interestingly, VU0453595 fully restored impaired LTD as well as deficits in cognitive function and social interaction in these mice. These results provide critical new insights into synaptic changes that may contribute to behavioral deficits in this mouse model and support a role for selective M1 PAMs as a novel approach for the treatment of schizophrenia.

Crystal structure of RNase A complexed with d(pA)4.

Co-crystals of pancreatic RNase A complexed with oligomers of d(pA)4 were grown from polyethylene glycol 4000 at low ionic strength and the X-ray diffraction data were collected to 2.5 A resolution. From a series of heavy-atom derivatives a multiple isomorphous replacement-phased electron density map of the RNase-d(pA)4 complex was calculated to 3.5 A. By inspection, the disposition of the known structure of RNase in the unit cell was determined and this was confirmed by calculation of a standard crystallographic residual, R. Refinement of the protein alone in the unit cell as a strictly rigid body yielded an R factor of 0.32 at 2.8 A resolution. From difference Fourier syntheses DNA fragments were elucidated and incorporated into a model of the complex. The entire asymmetric unit was refined using a restrained-constrained least-squares procedure (CORELS) interspersed with difference Fourier syntheses. At the present time the crystal structure has been refined to an overall R value of 0.215 at 2.5 A resolution. The asymmetric unit of the complex crystals contains four oligomers of d(pA)4 associated with each molecule of RNase. In addition, there may also be partially ordered fragments of DNA at low occupancy present in the unit cell, but these have not, at this time, been incorporated into the model. One tetramer of d(pA)4 is entirely bound by a single protein molecule and occupies a portion of the active site cleft, filling the purine binding site and the phosphate site at the catalytic center with its 5' nucleotide. Two other tetramers are partly intermolecular. One passes from near the pyrimidine binding site over the surface of the protein toward arginine 39 and into a solvent region. A third tetramer is anchored at its 5' terminus by a salt link to lysine 98, passes near arginine and then through a solvent region to terminate with its 3' end near the surface of another protein molecule in the lattice. The fourth tetramer of d(pA)4 is bound at its 5' end on the opposite side of the protein from the active site in an electropositive anion trap that includes lysines 31 and 91 as well as arginine 33. There may be a DNA-DNA interaction involving the 5' phosphate of one tetramer and the 3' bases of two other tetramers and this may help to stabilize the crystalline complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Solution structures of the rabbit neutrophil defensin NP-5.

Solution structures of the rabbit neutrophil defensin NP-5 have been determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and distance geometry techniques. This 33 amino acid peptide is part of the oxygen-independent mammalian defense system against microbial infection. The structures were generated from 107 n.m.r. derived inter-residue proton-proton distance constraints. A distance geometry algorithm was then used to determine the range of structures consistent with these distance constraints. These distance geometry calculations employed an improved algorithm that allowed the chirality constraints to be relaxed on prochiral centers when it was not possible to make stereo-specific assignments of protons on these centers. This procedure gave superior results compared with standard distance geometry methods and also produced structures that were more consistent with the original n.m.r. data. Analysis of the NP-5 structures shows that the overall folding of the peptide backbone is well defined by the n.m.r. distance information but that the side-chain group conformations are generally less well defined.

Milk and lactose-hydrolyzed milk.

Seven females 18 to 26 years old (mean 23 years) who were lactose malabsorbers as determined by a lactose tolerance test were given two different test meals: 5 ml/kg body weight reconstituted nonfat dry milk and 5 ml/kg body weight reconsituted nonfat dry milk in which 92% of the lactose had been hydrolyzed to glucose and galactose. Lactose malabsorption was determined by the breath H2 test. Tests were done in duplicate for a total of 28 tests. Whether a 3-hr test period of a 5-hr test period was used, the average breath H2 response after the lactose-hydrolyzed nonfat dry milk test meals was significantly lower (P less than 0.002) than the average breath H2 response after the nonfat dry milk test meals. There were significant differences among individual responses (P lless than 0.06 for the 3-hr test period and P less than 0.09 for the 5-hr test period), and no significant differences between duplicate test days.

The determination of carbonic anhydrase-2 phenotypes in dried bloodstains by cellulose acetate electrophoresis.

A new, simple, and rapid method for phenotyping carbonic anhydrase-2 (CA2) on bloodstains 15 weeks old was performed. Separation of the CA2 isozymes was accomplished by cellulose acetate electrophoresis. The staining substrate was fluorescein diacetate.

Sites of attachment and density assessment in Amblyomma americanum (Acari: Ixodidae) on nursing beef calves.

Primary attachment sites of male, female, and nymphal Amblyomma americanum (L.) on nursing beef calves were the axillary, inguinal and perianal areas of the body. About 50% of all larvae fed on the ears. A density assessment method which uses the number of A. americanum on less than or equal to 5 body areas to predict the total number of ticks (tick burden) on the left side of a calf is presented. Estimates of tick burden for male, female, nymphal and larval A. americanum each require examination of the axillary, inguinal, and perianal body areas. In addition, to estimate larval and male A. americanum, the neck, dewlap and brisket require examination as do the ears for estimates of nymphs and larvae. Mean tick burden in a herd of calves can be estimated by an examination of from 14 to 100% of all calves depending on the level of precision required for the estimate.

Interval sampling of breath hydrogen (H2) as an index of lactose malabsorption in lactase-deficient subjects.

Interval sampling of breath hydrogen content was used in lactose malabsorbers: (1) to compare hydrogen responses following increasing oral doses of lactose in milk and aqueous solutions; (2) to determine the reproducibility of interval breath sampling, and (3) to compare carbohydrate malabsorption following ingestion of either regular milk or milk containing Lactobacillus acidophilus. Significant differences in breath hydrogen responses due to increasing amounts of lactose in milk and aqueous solutions were observed. The individual breath hydrogen responses were reproducible using the same lactose dose on different days. There was no significant difference in breath hydrogen responses or symptoms following administration of either regular milk or milk containing Lactobacillus acidophilus. Breath hydrogen sampling at intervals, as performed in these studies, provides a sensitive and reproducible index of lactose malabsorption.