Search

1-Phenyl-8-azabicyclo[3.2.1]octane ethers are NK(1) receptor antagonists. Substitution at the 6-exo-position led to high affinity NK(1) antagonists with a prolonged duration of action in vivo. Incorporation of an alpha-methyl substituent in the pendent benzyl ether side chain gave compounds with increased selectivity over the hERG channel.

The human organic anion transporter, OAT3 (SLC22A8), plays a critical role in renal drug elimination, by mediating the entry of a wide variety of organic anions, including a number of commonly used pharmaceuticals, into the renal proximal tubular cells. To understand the nature and extent of genetic variation in OAT3, and to determine whether such variation affects its function, we identified OAT3 variants in a large, ethnically diverse sample population and studied their transport activities in cellular assays. We identified a total of 10 distinct coding-region variants, which altered the encoded amino acid sequence, in DNA samples from 270 individuals (80 African-Americans, 80 European-Americans, 60 Asian-Americans, and 50 Mexican-Americans). The overall prevalence of these OAT3 variants was relatively low among the screened population, with only three variants having allele frequencies of >1% in a particular ethnic group. Clones of each variant were created by site-directed mutagenesis, expressed in HEK-293 cells, and tested for function using the model substrates, estrone sulfate (ES) and cimetidine (CIM). The results revealed a high degree of functional heterogeneity among OAT3 variants, with three variants (p. Arg149Ser, p. Gln239Stop, and p. Ile260Arg) that resulted in complete loss of function, and several others with significantly reduced function. One of the more common variants (p. Ile305Phe), found in 3.5% of Asian-Americans, appeared to have altered substrate specificity. This variant exhibited a reduced ability to transport ES, but a preserved ability to transport CIM. These data suggest that genetic variation in OAT3 may contribute to variation in the disposition of drugs.

Background: Elongation-induced vertebral artery injury has been hypothesized to occur during non-physiological coupled axial rotation and extension of head. No studies have quantified dynamic vertebral artery elongation during head-turned rear impacts. Therefore, we evaluated effect of rotated head posture vs. forward head posture at the time of impact on dynamic vertebral artery elongation during simulated rear impacts., Methods: A whole cervical spine model with surrogate head and muscle force replication underwent either simulated head-turned (n = 6) or head-forward (n = 6) rear impacts of 3.5, 5, 6.5 and 8 g. Continuous dynamic vertebral artery elongation was recorded using custom transducer and compared to physiological values obtained during intact flexibility testing., Findings: Average (SD) peak dynamic vertebral artery elongation of up to 30.5 (2.6) mm during head-turned rear-impact significantly exceeded (P < 0.05) the physiological beginning at 5 g. Highest peak elongation of 5.8 (2.1) mm during head-forward rear impact did not exceed physiological limit. Head-turned rear impact caused earlier occurrence of average peak vertebral artery elongation, 84.5 (4.2) ms vs. 161.0 (43.8) ms, and higher average peak vertebral artery elongation rate, 1336.7 (74.5) mm/s vs. 211.5 (97.4) mm/s, as compared to head-forward rear impact., Interpretation: Elongation-induced vertebral artery injury is more likely to occur in those with rotated head posture at the time of rear impact, as compared to head-forward.

A series of 4,4-disubstituted cyclohexylamine NK(1) antagonists containing a lactam ring is described. The compounds are brain penetrant and activity is demonstrated in a ferret emesis model.

The 26S proteasome is the primary protease responsible for degrading misfolded membrane proteins in the endoplasmic reticulum. Here we examine the specific role of beta subunit function on polypeptide cleavage and membrane release of CFTR, a prototypical ER-associated degradation substrate with 12 transmembrane segments. In the presence of ATP, cytosol and fully active proteasomes, CFTR was rapidly degraded and released into the cytosol solely in the form of trichloroacetic acid (TCA)-soluble peptide fragments. Inhibition of proteasome beta subunits markedly decreased CFTR degradation but surprisingly, had relatively minor effects on membrane extraction and release. As a result, large TCA-insoluble degradation intermediates derived from multiple CFTR domains accumulated in the cytosol where they remained stably bound to inhibited proteasomes. Production of TCA-insoluble fragments varied for different proteasome inhibitors and correlated inversely with the cumulative proteolytic activities of beta1, beta2 and beta5 subunits. By contrast, ATPase inhibition decreased CFTR release but had no effect on the TCA solubility of the released fragments. Our results indicate that the physiologic balance between membrane extraction and peptide cleavage is maintained by excess proteolytic capacity of the 20S subunit. Active site inhibitors reduce this capacity, uncouple ATPase and peptidase activities, and generate cytosolic degradation intermediates by allowing the rate of unfolding to exceed the rate of polypeptide cleavage.

Background: Linkage studies in autism have identified susceptibility loci on chromosomes 2q and 7q, regions containing the DLX1/2 and DLX5/6 bigene clusters. The DLX genes encode homeodomain transcription factors that control craniofacial patterning and differentiation and survival of forebrain inhibitory neurons. We investigated the role that sequence variants in DLX genes play in autism by in-depth resequencing of these genes in 161 autism probands from the AGRE collection., Results: Sequencing of exons, exon/intron boundaries and known enhancers of DLX1, 2, 5 and 6 identified several nonsynonymous variants in DLX2 and DLX5 and a variant in a DLX5/6 intragenic enhancer. The nonsynonymous variants were detected in 4 of 95 families from which samples were sequenced. Two of these four SNPs were not observed in 378 undiagnosed samples from North American populations, while the remaining 2 were seen in one sample each., Conclusion: Segregation of these variants in pedigrees did not generally support a contribution to autism susceptibility by these genes, although functional analyses may provide insight into the biological understanding of these important proteins.

Objective: To evaluate the impact of a smoking-cessation media campaign for teens on utilization of a cessation Web site, GottaQuit.com., Methods: Telephone surveys were conducted before and after the implementation of a countywide media campaign to promote the use of a smoking-cessation Web site for youths. The surveys were designed to assess teen awareness and utilization of the Web site, as well as tobacco use and cessation attempts. Supplemental 2003 Youth Risk Behavior Survey items also assessed use of the Web site., Results: Most teen smokers reported that they wanted to quit smoking. Almost all teens reported exposure to GottaQuit.com ads and accurately identified GottaQuit.com as a Web site that offers cessation help for youths. Nearly 1 in 4 smokers who were trying to quit had visited GottaQuit.com or another Web site for cessation assistance., Conclusions: The GottaQuit.com campaign effectively reached almost all teens, regardless of smoking status. Smokers were more likely than nonsmokers to have visited the Web site for help with quitting. Web adjuncts are likely to be used by adolescents who seek assistance in quitting.

Objectives: The organic anion transporter, OAT1 (SLC22A6), plays a role in the renal elimination of many drugs and environmental toxins. The goal of this study was to identify and functionally characterize OAT1 variants as a first step towards understanding whether genetic variation in OAT1 may contribute to interindividual differences in renal elimination of xenobiotics., Methods: As part of a larger study, 276 DNA samples from an ethnically diverse population were screened and 12 coding region variants of OAT1 were identified. The non-synonymous variants were then constructed and characterized in Xenopus laevis oocytes. A small family-based clinical study was conducted to determine the renal elimination of a model OAT1 substrate, adefovir (an antiviral agent) in human subjects who possessed a non-functional variant, OAT1-R454Q., Results: Six non-synonymous variants were identified; two (OAT1-R50 H and OAT1-R293W) were present at > or = 1% in at least one ethnic population. These two variants exhibited normal uptake of p-aminohippurate, ochratoxin A and methotrexate assayed in X. laevis oocytes. One variant, OAT1-R454Q, was non-functional with respect to the above substrates. In the clinical study, there was no significant decrease in the renal secretory clearance of adefovir in family members heterozygous for OAT1-454Q in comparison to those with the reference transporter, OAT1-454R., Conclusions: These data indicate that the coding region of OAT1 has low genetic and functional diversity and suggest that coding region variants of OAT1 may not contribute substantially to interindividual differences in renal elimination of xenobiotics.

The concentrative nucleoside transporter CNT2 (SPNT1; SLC28A2) plays a role in the absorption and disposition of naturally occurring nucleosides, as well as nucleoside analog drugs. The aim of the present study was to characterize genetic variation in SLC28A2, the gene encoding CNT2, and to functionally analyse non-synonymous variants of CNT2, as a first step towards understanding whether genetic variation in this nucleoside transporter contributes to variation in response to nucleoside analogs. As part of a larger study, DNA samples from an ethnically diverse population (100 African-Americans, 100 European-Americans, 30 Asians, 10 Mexicans and seven Pacific Islanders) were screened and 10 coding region variants of CNT2 were identified. The non-synonymous variants were then constructed and characterized in Xenopus laevis oocytes. Six non-synonymous variants were identified, and all were able to transport guanosine. The four common variants (>1% in the sample population) were further characterized with the anti-viral nucleoside analog drug ribavirin. No differences were observed among the four common variants in the uptake kinetics of 3H-ribavirin (Km in microM: 35.6+/-9.27 for CNT2-reference, 40.7+/-6.47 for CNT2-P22L, 31.2+/-15.8 for CNT2-S75R, 26.7+/-6.13 for CNT2-S245T and 49.9+/-14.6 for CNT2-F355S). The variant CNT2-F355S exhibited a change in specificity for the naturally occurring nucleosides, inosine and uridine. All non-synonymous variants of CNT2 took up guanosine, and the four variants examined showed no significant difference in ribavirin kinetics. However, CNT2-F355S (3% allele frequency in the African-American sample) was found to alter specificity for naturally occurring nucleosides, which may have implications for nucleoside homeostasis.

The human concentrative nucleoside transporter, CNT3 (SLC28A3), plays an important role in mediating the cellular entry of a broad array of physiological nucleosides and synthetic anticancer nucleoside analog drugs. As a first step toward understanding the genetic basis for interindividual differences in the disposition and response to antileukemic nucleoside analogs, we examined the genetic and functional diversity of CNT3. In all, 56 variable sites in the exons and flanking intronic region of SLC28A3 were identified in a collection of 270 DNA samples from US populations (80 African-Americans, 80 European-Americans, 60 Asian-Americans, and 50 Mexican-Americans). Of the 16 coding region variants, 12 had not been previously reported. Also, 10 resulted in amino-acid changes and three of these had total allele frequencies of >/=1%. Nucleotide diversity (pi) at nonsynonymous and synonymous sites was estimated to be 1.81 x 10(4) and 18.13 x 10(4), respectively, suggesting that SLC28A3 is under negative selection. All nonsynonymous variants, constructed by site-directed mutagenesis and expressed in Xenopus laevis oocytes, transported purine and pyrimidine model substrates, except for c. 1099G>A (p. Gly367Arg). This rare variant alters an evolutionarily conserved site in the putative substrate recognition domain of CNT3. The presence of three additional evolutionarily conserved glycine residues in the vicinity of p. Gly367Arg that are also conserved in human paralogs suggest that these glycine residues are critical in the function of the concentrative nucleoside transporter family. The genetic analysis and functional characterization of CNT3 variants suggest that this transporter does not tolerate nonsynonymous changes and is important for human fitness.

Two mouse models are widely used for Down syndrome (DS) research. The Ts65Dn mouse carries a small chromosome derived primarily from mouse chromosome 16, causing dosage imbalance for approximately half of human chromosome 21 orthologs. These mice have cerebellar pathology with direct parallels to DS. The Ts1Cje mouse, containing a translocated chromosome 16, is at dosage imbalance for 67% of the genes triplicated in Ts65Dn. We quantified cerebellar volume and granule cell and Purkinje cell density in Ts1Cje. Cerebellar volume was significantly affected to the same degree in Ts1Cje and Ts65Dn, despite that Ts1Cje has fewer triplicated genes. However, dosage imbalance in Ts1Cje had little effect on granule cell and Purkinje cell density. Several mice with dosage imbalance for the segment of the Ts65Dn chromosome not triplicated in Ts1Cje had phenotypes that contrasted with those in Ts1Cje. These observations do not readily differentiate between two prevalent hypotheses for gene action in DS., (Copyright 2004 Wiley-Liss, Inc.)

The concentrative nucleoside transporter, CNT1 (SLC28A1), mediates the cellular uptake of naturally occurring pyrimidine nucleosides and many structurally diverse anticancer and antiviral nucleoside analogs. As a first step toward understanding whether genetic variation in CNT1 contributes to variation in the uptake and disposition of clinically used nucleoside analogs, we determined the haplotype structure and functionally analyzed all coding region variants of CNT1 identified in ethnically diverse populations (100 African Americans, 100 European Americans, 30 Asians, 10 Mexican Americans, and 7 Pacific Islanders) (Leabman et al., 2003). A total of 58 coding region haplotypes were identified using PHASE analysis, 44 of which contained at least one amino acid variant. More than half of the coding region haplotypes were population-specific. Using site-directed mutagenesis, 15 protein-altering CNT1 variants, including one amino acid insertion and one base pair (bp) deletion, were constructed and expressed in Xenopus laevis oocytes. All variant transporters took up [3H]thymidine with the exception of CNT1-Ser546Pro, a rare variant, and CNT1-1153del, a single bp deletion found at a frequency of 3% in the African American population. The bp deletion results in a frame-shift followed by a stop-codon. The anticancer nucleoside analog gemcitabine had a reduced affinity for CNT1-Val189Ile (a common CNT1 variant found at a frequency of 26%) compared with reference CNT1 (IC50=13.8 +/- 0.60 microM for CNT1-reference and 23.3 +/- 1.5 microM for CNT1-Val189Ile, p<0.05). These data suggest that common genetic variants of CNT1 may contribute to variation in systemic and intracellular levels of anti-cancer nucleoside analogs.

Objectives: There is increasing evidence that polymorphism of the ABCB1 (MDR1) gene contributes to interindividual variability in bioavailability and tissue distribution of P-glycoprotein substrates. The aim of the present study was to (1) identify and describe novel variants in the ABCB1 gene, (2) understand the extent of variation in ABCB1 at the population level, (3) analyze how variation in ABCB1 is structured in haplotypes, and (4) functionally characterize the effect of the most common amino acid change in P-glycoprotein., Methods and Results: Forty-eight variant sites, including 30 novel variants and 13 coding for amino acid changes, were identified in a collection of 247 ethnically diverse DNA samples. These variants comprised 64 statistically inferred haplotypes, 33 of which accounted for 92% of chromosomes analyzed. The two most common haplotypes, ABCB1*1 and ABCB1*13, differed at six sites (three intronic, two synonymous, and one non-synonymous) and were present in 36% of all chromosomes. Significant population substructure was detected at both the nucleotide and haplotype level. Linkage disequilibrium was significant across the entire ABCB1 gene, especially between the variant sites found in ABCB1*13, and recombination was inferred. The Ala893Ser change found in the common ABCB1*13 haplotype did not affect P-glycoprotein function., Conclusion: This study represents a comprehensive analysis of ABCB1 nucleotide diversity and haplotype structure in different populations and illustrates the importance of haplotype considerations in characterizing the functional consequences of ABCB1 polymorphisms.

Extensive screening of compound libraries was undertaken to identify compounds with high affinity for the rat NK(1) receptor based on inhibition of [(125)I]-substance P binding. RP67580, SR140333, NKP-608 and GR205171 were selected as compounds of interest, with cloned rat NK(1) receptor binding K(i) values of 0.15-1.9 nM. Despite their high binding affinity, NKP-608 and GR205171 exhibited only a moderate functional antagonism of substance P-induced inositol-1-phosphate accumulation and acidification rate at 1 microM using cloned or native rat NK(1) receptors in vitro. The ability of the compounds to penetrate the CNS was determined by inhibition of NK(1) agonist-induced behaviours in gerbils and rats. GR205171 and NKP-608 potently inhibited GR73632-induced foot drumming in gerbils (ID(50) 0.04 and 0.2 mg/kg i.v., respectively). In contrast, RP67580 and SR140333 were poorly brain penetrant in gerbils (no inhibition at 10 mg/kg i.v.) and were not examined further in vivo. In rats, only high doses of GR205171 (10 or 30 mg/kg s.c.) inhibited NK(1) agonist-induced sniffing and hypertension, whilst NKP-608 (1 or 10 mg/kg i.p.) was without effect. GR205171 (3-30 mg/kg s.c.) caused only partial inhibition of separation-induced vocalisations in rat pups, a response that is known to be NK(1) receptor mediated in other species. These observations demonstrate the shortcomings of currently available NK(1) receptor antagonists for rat psychopharmacology assays.

Altered neuronal endocytosis is the earliest known pathology in sporadic Alzheimer's disease (AD) and Down syndrome (DS) brain and has been linked to increased Abeta production. Here, we show that a genetic model of DS (trisomy 21), the segmental trisomy 16 mouse Ts65Dn, develops enlarged neuronal early endosomes, increased immunoreactivity for markers of endosome fusion (rab5, early endosomal antigen 1, and rabaptin5), and endosome recycling (rab4) similar to those in AD and DS individuals. These abnormalities are most prominent in neurons of the basal forebrain, which later develop aging-related atrophy and degenerative changes, as in AD and DS. We also show that App, one of the triplicated genes in Ts65Dn mice and human DS, is critical to the development of these endocytic abnormalities. Selectively deleting one copy of App or a small portion of the chromosome 16 segment containing App from Ts65Dn mice eliminated the endosomal phenotype. Overexpressing App at high levels in mice did not alter early endosomes, implying that one or more additional genes on the triplicated segment of chromosome 16 are also required for the Ts65Dn endosomal phenotype. These results identify an essential role for App gene triplication in causing AD-related endosomal abnormalities and further establish the pathogenic significance of endosomal dysfunction in AD.

Membrane transporters maintain cellular and organismal homeostasis by importing nutrients and exporting toxic compounds. Transporters also play a crucial role in drug response, serving as drug targets and setting drug levels. As part of a pharmacogenetics project, we screened exons and flanking intronic regions for variation in a set of 24 membrane transporter genes (96 kb; 57% coding) in 247 DNA samples from ethnically diverse populations. We identified 680 single nucleotide polymorphisms (SNPs), of which 175 were synonymous and 155 caused amino acid changes, and 29 small insertions and deletions. Amino acid diversity (pi(NS)) in transmembrane domains (TMDs) was significantly lower than in loop domains, suggesting that TMDs have special functional constraints. This difference was especially striking in the ATP-binding cassette superfamily and did not parallel evolutionary conservation: there was little variation in the TMDs, even in evolutionarily unconserved residues. We used allele frequency distribution to evaluate different scoring systems (Grantham, blosum62, SIFT, and evolutionarily conservedevolutionarily unconserved) for their ability to predict which SNPs affect function. Our underlying assumption was that alleles that are functionally deleterious will be selected against and thus under represented at high frequencies and over represented at low frequencies. We found that evolutionary conservation of orthologous sequences, as assessed by evolutionarily conservedevolutionarily unconserved and SIFT, was the best predictor of allele frequency distribution and hence of function. European Americans had an excess of high frequency alleles in comparison to African Americans, consistent with a historic bottleneck. In addition, African Americans exhibited a much higher frequency of population specific medium-frequency alleles than did European Americans.

K-edge X-ray absorption and EPR spectroscopies were used to test the variation in blood cell vanadium between and within specimens of the tunicate Ascidia ceratodes from Bodega Bay, California. Intracellular vanadium was speciated by fitting the XAS spectra of whole blood cells with linear combinations of the XAS spectra of models. Blood cell samples representing one specimen each, respectively, revealed 92.5 and 38.7% of endogenous vanadium as [V(H(2)O)(6)](3+), indicating dissimilar distributions. Conversely, vanadium distributions within blood cell samples respectively representing one and six specimens proved very similar. The derived array of V(III) complexes was consistent with multiple intracellular regions that differ both in pH and c(sulfate), both within and between specimens. No systematic effect on vanadium distribution was apparent on mixing blood cells. EPR and XAS results indicated at least three forms of endogenous vanadyl ion, two of which may be dimeric. An inverse linear correlation was found between soluble and complexed forms of vanadyl ion, implying co-regulation. The EPR A value of endogenous vanadyl ion [A(0)=(1.062+/-0.008)x10(-2) cm(-1)] was marginally different from that representing Monterey Bay A. ceratodes [A(0)=(1.092+/-0.006) x10(-2) cm(-1)]. Comparisons indicate that Bodega Bay A. ceratodes maintain V(III) in a more acidic intracellular environment on average than do those from Monterey Bay, showing variation across populations. Blood cell vanadium thus noticeably diverges at all organismal levels among A. ceratodes.

The multidisciplinary UCSF Pharmacogenetics of Membrane Transporters project seeks to systematically identify sequence variants in transporters and to determine the functional significance of these variants through evaluation of relevant cellular and clinical phenotypes. The project is structured around four interacting cores: genomics, cellular phenotyping, clinical phenotyping, and bioinformatics. The bioinformatics core is responsible for collecting, storing, and analyzing the information obtained by the other cores and for presenting the results, in particular, for the genomic data. Most of this process is automated using locally developed software written in Python, an open source language well suited for rapid, modular development that meets requirements that are themselves constantly evolving. Here we present the details of transforming ABI trace file data into useful information for project investigators and a description of the types of data analysis and display that we have developed.

Objective: To evaluate in a prospective, controlled fashion the prevalence of umbilical artery Doppler waveform notching and its association with cord and placental abnormalities., Methods: During a 6-month period, umbilical artery velocity waveforms were prospectively obtained on 1857 pregnancies at greater than 27 weeks' gestation. All pregnant patients with the presence of a persistent fetal umbilical artery waveform notch formed the study population (cases). Control patients, matched for gestational age, with normal umbilical artery waveforms, were selected for comparison (2 controls per case). After delivery, detailed pathologic examination was performed on all umbilical cords and placentas., Results: The presence of an umbilical artery waveform notch was noted in 29 (1.6%) of 1857 pregnancies. Postnatal placental evaluation showed the presence of an accessory placental lobe in 5 (17%) of 29 cases compared with 1 (1.8%) of 54 controls (P = .018). Overall, the presence of an umbilical artery waveform notch was associated with umbilical cord abnormalities in 21 (72%) of 29 cases compared with 8 (14%) of 54 controls (odds ratio, 15; 95% confidence interval, 4.4-54.4)., Conclusions: Umbilical artery waveform notching appears to be a strong predictor of cord and placental abnormalities. This finding may have important clinical implications.

A series of novel 4,4-disubstituted cyclohexylamines as NK(1) receptor antagonists is described: modifications to the amine moiety retain NK(1) receptor binding affinity whilst disrupting I(Kr) affinity.

Trisomy for chromosome 21 (Chr 21) has profound effects on development that result in a constellation of phenotypes known as Down syndrome (DS). Distinctive craniofacial manifestations are among the few features common to all individuals with DS. The characteristic face of a person with DS results primarily from maldevelopment of the underlying craniofacial skeleton. The Ts65Dn mouse, which has segmental trisomy 16, producing dosage imbalance for about half the genes found on human Chr 21, exhibits specific skeletal malformations corresponding directly to the craniofacial dysmorphogenesis in DS. Here we demonstrate that Ts1Cje mice, which are at dosage imbalance for about 3/4 of the genes triplicated in Ts65Dn, demonstrate a very similar pattern of anomalies in the craniofacial skeleton. However, one characteristic of Ts65Dn mice, a broadening of the cranial vault contributing to brachycephaly, is not seen in Ts1Cje mice. These observations independently confirm that a dosage imbalance for mouse genes orthologous to those on human Chr 21 has corresponding effects in both species. The subtle differences in the craniofacial phenotypes of Ts1Cje and Ts65Dn mice have implications for elucidation of the mechanisms by which this aneuploidy disrupts development., (Copyright 2001 Wiley-Liss, Inc.)

1-(5-[[(2R,3S)-2-([(1R)-1-[3,5-Bis(trifluoromethyl)phenyl]ethyl]oxy)-3-(4-fluorophenyl)morpholin-4-yl]methyl]-2H-1,2,3-triazol-4-yl)-N,N-dimethylmethanamine hydrochloride 3 is a high affinity, orally active, h-NK(1) receptor antagonist with a long central duration of action and a solubility in water of >100 mg/mL. The construction of the 5-dimethylaminomethyl 1,2,3-triazol-4-yl unit, which incorporates the solubilizing group of 3, was accomplished by thermal rearrangement of a propargylic azide in the presence of dimethylamine. Compound 3 is highly effective in pre-clinical tests that are relevant to clinical efficacy in emesis and depression.

Misfolded proteins in the endoplasmic reticulum (ER) are degraded by N-terminal threonine proteases within the 26S proteasome. Each protease is formed by an activated beta subunit, beta5/X, beta1/Y, or beta2/Z, that exhibits chymotrypsin-like, peptidylglutamyl-peptide hydrolyzing, or trypsin-like activity, respectively. Little is known about the relative contribution of specific beta subunits in the degradation of endogenous protein substrates. Using active site proteasome inhibitors and a reconstituted degradation system, we now show that all three active beta subunits can independently contribute to ER-associated degradation of the cystic fibrosis transmembrane conductance regulator (CFTR). Complete inactivation (>99.5%) of the beta5/X subunit decreased the rate of ATP-dependent conversion of CFTR to trichloroacetic acid soluble fragments by only 40%. Similarly, proteasomes containing only active beta1/Y or beta2/Z subunits degraded CFTR at approximately 50% of the rate observed for fully functional proteasomes. Simultaneous inhibition (>93%) of all three beta subunits blocked CFTR degradation by approximately 90%, and inhibition of both protease and ATPase activities was required to completely prevent generation of small peptide fragments. Our results demonstrate both a conserved hierarchy (ChT-L > PGPH > or = T-L) as well as a redundancy of beta subunit function and provide insight into the mechanism by which active site proteasome inhibitors influence degradation of endogenous protein substrates at the ER membrane.

The phenotype of NK1R-/- mice was compared with that of acute pharmacological blockade of the tachykinin NK1 receptor on sensorimotor function and in assays relevant to depressive illness and anxiety. The dose range for L-760735 and GR205171 that was associated with functional blockade of central NK1 receptors in the target species was established by antagonism of the behavioural effects of intracerebroventricular NK1 agonist challenge in gerbils, mice and rats. The caudal grooming and scratching response to GR73632 was absent in NK1R-/- mice, confirming that the receptor had been genetically ablated. There was no evidence of sedation or motor impairment in NK1R-/- mice or following administration of L-760735 to gerbils, even at doses in excess of those required for central NK1 receptor occupancy. In the resident-intruder and forced swim test, the behaviour of NK1R-/- mice, or animals treated acutely with L-760735 or GR205171, resembled that seen with the clinically used antidepressant drug fluoxetine. However, the effects of GR205171 were not clearly enantioselective in mice. In contrast, although NK1R-/- mice also exhibited an increase in the duration of struggle behaviour in the tail suspension test, this was not observed following pharmacological blockade with L-760735 in gerbils or GR205171 in mice, suggesting that this may reflect a developmental alteration in the knockout mouse. There was no effect of NK1 receptor blockade with L-760735 in guinea-pigs or GR205171 in rats, or deletion of the NK1 receptor in mice, on behaviour in the elevated plus-maze test for anxiolytic activity. These findings extend previous observations on the phenotype of the NK1R-/- mouse and establish a broadly similar profile following acute pharmacological blockade of the receptor. These studies also serve to underscore the limitations of currently available antagonists that are suitable for use in rat and mouse behavioural assays.

Mn superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme, has been shown to be essential for animal survival. MnSOD mutant mice (Sod2-/- mice) on the CD1 background develop severe dilated cardiomyopathy and usually die within 10 d after birth. To characterize better the phenotype and understand the mechanism of superoxide-mediated tissue damage in Sod2-/- mice, congenic Sod2-/- mice on inbred backgrounds were generated to ensure genetic homogeneity. When generated on a C57BL/6J background (B6), more than half of the fetuses develop severe dilated cardiomyopathy by embryonic day 15 and die in the uterus. Those that survive to term usually die within 24 h. In contrast, Sod2-/- mice on DBA/2J (D2) and B6D2F1 (B6D2F1) backgrounds develop normally throughout gestation and do not develop dilated cardiomyopathy. However, the D2 mice do develop a severe metabolic acidosis and survive for only up to 12 d after birth. B6D2F1) mice have a milder form of metabolic acidosis and can survive for up to 3 weeks. The marked difference in lifespans and the development of dilated cardiomyopathy in the B6 but not the D2 or B6D2F1 backgrounds indicate the possible existence of genetic modifiers that provide protection to the developing hearts in the absence of MnSOD.

Manganese superoxide dismutase (MnSOD) is essential in protecting mitochondria against the damaging effects of superoxide radicals (O(2)(*-)), and increased expression of MnSOD protects cells and transgenic animals from various forms of oxidative stress. In addition, increased levels of MnSOD have been shown to slow down cell growth and induce differentiation. To study the effects of high MnSOD levels in vivo, we generated a series of transgenic mice using a mouse genomic sequence under control of the endogenous promoter. Four transgenic lines produced by pronuclear DNA injection exhibited up to 2-fold elevated MnSOD levels in brain and heart. However, using an embryonic stem cell approach, a line having 10-fold elevated MnSOD levels in the brain and 6- to 7-fold elevated levels in the heart and kidney was generated. Surprisingly, the genetic background of this transgenic line influenced the expression level of the transgene, with DBA/2 (D2) and C57BL/6 (B6) mice exhibiting low- and high-level transgene expression, respectively. This difference was the result of an increased transcription rate of the transgene. High-level MnSOD expression in B6 animals was associated with small size, male infertility, and decreased female fertility. These features are absent on the D2 background and indicate that high levels of MnSOD activity may interfere with normal growth and fertility.

Heart mitochondria from heterozygous (Sod2(-/+)) knockout mice have a 50% reduction in manganese superoxide dismutase (MnSOD) activity. The decrease in MnSOD activity was associated with increased mitochondrial oxidative damage as demonstrated by a decrease in the activities of iron sulfhydryl proteins sensitive to oxygen stress (aconitase and reduced nicotinamide adenine dinucleotide-oxidoreductase). Mitochondrial function was altered in the Sod2(-/+) mice, as shown by decreased respiration by complex I and an increase in the sensitivity of the permeability transition to induction by calcium and t-butylhydroperoxide. The increased induction of the permeability transition in heart mitochondria from Sod2(-/+.)mice was associated with increased release of cytochrome c and an increase in DNA fragmentation. Cardiomyocytes isolated from neonatal Sod2(-/+) and Sod2(-/-) mice were more sensitive to cell death than cardiomyocytes from Sod2(+/+) mice after t-butylhydroperoxide treatment, and this increased sensitivity was prevented by inhibiting the permeability transition with cyclosporin A. These experiments demonstrate that MnSOD may play an important role in the induction of the mitochondrial pathway of apoptosis in the heart, and this appears to occur primarily through the permeability transition.

Age-related degeneration of basal forebrain cholinergic neurons (BFCNs) contributes to cognitive decline in Alzheimer's disease and Down's syndrome. With aging, the partial trisomy 16 (Ts65Dn) mouse model of Down's syndrome exhibited reductions in BFCN size and number and regressive changes in the hippocampal terminal fields of these neurons with respect to diploid controls. The changes were associated with significantly impaired retrograde transport of nerve growth factor (NGF) from the hippocampus to the basal forebrain. Intracerebroventricular NGF infusion reversed well established abnormalities in BFCN size and number and restored the deficit in cholinergic innervation. The findings are evidence that even BFCNs chronically deprived of endogenous NGF respond to an intervention that compensates for defective retrograde transport. We suggest that age-related cholinergic neurodegeneration may be a treatable disorder of failed retrograde NGF signaling.

Mice inheriting both copies of MMU12 either maternally or paternally demonstrate imprinting effects. Whereas maternal uniparental disomy 12 (matUPD12) fetuses are growth retarded and die perinatally, paternal UPD12 (patUPD12) fetuses die during late gestation and exhibit placentomegaly and skeletal muscle maturation defects. To examine further the developmental consequences of UPD12, we intercrossed mouse stocks heterozygous for Robertsonian translocation chromosomes (8.12) and (10.12). We report that at 13.5-14.5 dg patUPD12 hearts exhibit increased ventricular diameter, thinner, less compact myocardium, and deep intertrabecular recesses when compared to controls. These data provide evidence for cardiac failure, a lethal condition, and suggest a role for an imprinted gene(s) in normal heart development.

Mucinous colorectal cancers exhibit a characteristic set of molecular genetic alterations and may be derived from progenitor cells committed to the goblet cell lineage. Previously, we demonstrated that the MUC2 mucin gene promoter drives transgene reporter expression with high specificity in small intestinal goblet cells of transgenic mice. On the basis of these experiments, we reasoned that the MUC2 promoter could be used to drive SV40 T antigen (Tag) expression in the same cell type, decoupling them from their normal antiproliferative controls. A line of mice was established (MUCTag6) that expressed Tag in intestinal goblet cells as determined by RNA blot and immunohistochemical analysis. These goblet cells were markedly involuted however, most notably in the villi. Endogenous intestinal MUC2 message levels were reduced to about one third the normal level in these mice. However, absorptive cell lineage markers were comparable with nontransgenics. Bromodeoxyuridine-positive S-phase cells are limited to crypts in nontransgenic intestine but are present in both crypts and villi in MUCTag6. In contrast, mitotic cells were not present in the villi, indicating that MUCTag6 villi goblet cells do not progress into M phase. Apoptotic cells positive for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling were increased more than fourfold in MUCTag6 villi (P < 0.0001), and apoptotic goblet cells were evident. Electron microscopic examination of MUCTag6 intestinal villi revealed the presence of degraded cell remnants containing mucin goblets together with other cell debris, further indicating apoptosis of the goblet cell lineage. Thus, the expression of Tag in intestinal goblet cells releases them from normal antiproliferative controls, causing their inappropriate entry into S phase even after they transverse the crypt/villus junction. They do not, however, progress to M phase. Instead, they undergo apoptosis with a high degree of efficiency in S or G(2) phase. These experiments demonstrate that apoptosis effectively blocks inappropriate goblet cell proliferation in the intestine, supporting its proposed role as an antineoplastic mechanism.

Hereditary non-X-linked nephrogenic diabetes insipidus (NDI) is caused by mutations in the aquaporin-2 (AQP2) water channel. In transfected cells, the human disease-causing mutant AQP2-T126M is retained at the endoplasmic reticulum (ER) where it is functional and targetable to the plasma membrane with chemical chaperones. A mouse knock-in model of NDI was generated by targeted gene replacement using a Cre-loxP strategy. Along with T126M, mutations H122S, N124S, and A125T were introduced to preserve the consensus sequence for N-linked glycosylation found in human AQP2. Breeding of heterozygous mice yielded the expected Mendelian distribution with 26 homozygous mutant offspring of 99 live births. The mutant mice appeared normal at 2-3 days after birth but failed to thrive and generally died by day 6 if not given supplemental fluid. Urine/serum analysis showed a urinary concentrating defect with serum hyperosmolality and low urine osmolality that was not increased by a V2 vasopressin agonist. Northern blot analysis showed up-regulated AQP2-T126M transcripts of identical size to wild-type AQP2. Immunoblots showed complex glycosylation of wild-type AQP2 but mainly endoglycosidase H-sensitive core glycosylation of AQP2-T126M indicating ER-retention. Biochemical analysis revealed that the AQP2-T126M protein was resistant to detergent solubilization. Kidneys from mutant mice showed collecting duct dilatation, papillary atrophy, and unexpectedly, some plasma membrane AQP2 staining. The severe phenotype of the AQP2 mutant mice compared with that of mice lacking kidney water channels AQP1, AQP3, and AQP4 indicates a critical role for AQP2 in neonatal renal function in mice. Our results establish a mouse model of human autosomal NDI and provide the first in vivo biochemical data on a disease-causing AQP2 mutant.

There is substantial evidence implicating mitochondrial dysfunction and free radical generation as major mechanisms of neuronal death in neurodegenerative diseases. The major free radical scavenging enzyme in mitochondria is manganese superoxide dismutase (SOD2). In the present study we investigated the susceptibility of mice with a partial deficiency of SOD2 to the neurotoxins 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), 3-nitropropionic acid (3-NP), and malonate, which are commonly used animal models of Parkinson's and Huntington's disease. Heterozygous SOD2 knockout (SOD2(+/-)) mice showed no evidence of neuropathological or behavioral abnormalities at 2-4 months of age. Compared to littermate wild-type mice, mice with partial SOD2 deficiency showed increased vulnerability to dopamine depletion after systemic MPTP treatment and significantly larger striatal lesions produced by both 3-NP and malonate. SOD2(+/-) mice also showed an increased production of "hydroxyl" radicals after malonate injection measured with the salicylate hydroxyl radical trapping method. These results provide further evidence that reactive oxygen species play an important role in the neurotoxicity of MPTP, malonate, and 3-NP. These findings show that a subclinical deficiency in a free radical scavenging enzyme may act in concert with environmental toxins to produce selective neurodegeneration.

Expression of wild type HFE reduces the ferritin levels of cells in culture. In this report we demonstrate that the predominant hereditary hemochromatosis mutation, C282Y(2) HFE, does not reduce ferritin expression. However, the second mutation, H63D HFE, reduces ferritin expression to a level indistinguishable from cells expressing wild type HFE. Further, two HFE cytoplasmic domain mutations engineered to disrupt potential signal transduction, S335M and Y342C, were functionally indistinguishable from wild type HFE in this assay, as was soluble HFE. These results implicate a role for the interaction of HFE with the transferrin receptor in lowering cellular ferritin levels.

Two animal models of Down syndrome (human trisomy 21) with segmental trisomy for all (Ts65Dn) or part (Ts1Cje) of human chromosome 21-homologous region of mouse chromosome 16 have cognitive and behavioral abnormalities. To compare these trisomies directly and to assess the phenotypic contribution of the region of difference between them, Ts65Dn, Ts1Cje, and a new segmental trisomic (Ms1Ts65) for the region of difference (APP: to Sod1) have been generated as littermates and tested in parallel. Although the performance of Ts1Cje mice in the Morris water maze is similar to that of Ts65Dn mice, the reverse probe tests indicate that Ts65Dn is more severely affected. By contrast, the deficits of Ms1Ts65 mice are significantly less severe than those of Ts65Dn. Therefore, whereas triplication of Sod1 to Mx1 plays the major role in causing the abnormalities of Ts65Dn in the Morris water maze, imbalance of APP: to Sod1 also contributes to the poor performance. Ts65Dn mice are hyperactive and Ts1Cje mice are hypoactive; the activity of Ms1Ts65 mice is not significantly above normal. These findings indicate that genes in the Ms1Ts65 trisomic region must interact with others in the Ts1Cje region to produce hyperactivity in Ts65Dn mice.

Objective: The lymphocyte is a principal mediator of the inflammatory response, and lymphocyte depletion via apoptosis may be an important mechanism of modulating inflammation. Increased oxygen consumption occurs during sepsis and results in the generation of reactive oxygen species. Although reactive oxygen species initiate apoptosis in many biological systems, their role in controlling lymphocyte apoptosis during sepsis is unclear. The objective of this study was to better characterize the role of oxidative stress in precipitating lymphocyte apoptosis during sepsis and to specifically define the role of the CuZn superoxide dismutase (SOD) enzyme complex, a major antioxidant defense, in modulating this process., Design: Prospective, randomized, controlled study., Setting: Research laboratory at an academic medical center., Subjects: Mice that were either genetically normal or that were deficient in or overexpressed the enzyme CuZn SOD., Interventions: Mice from each genetic group were randomized to no manipulation (control), sham surgery, or cecal ligation and puncture. Mice were killed 18-24 hrs after study entry, and the thymi and spleen were removed for analysis of apoptosis., Measurements and Main Results: Lymphocyte apoptosis was assessed by three independent methods: light microscopy, fluorescent terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling, and DNA gel electrophoresis. Comparisons were performed using standard parametric statistical tests. Lymphocyte apoptosis was present in mice after CLP but not in control mice or in mice after sham surgery (p < .05). Mice completely lacking CuZn SOD developed significantly more lymphocyte apoptosis than did either partially CuZn SOD-deficient or genetically normal mice (p < .05). This apoptosis was more pronounced in the thymus than the spleen and, within the thymus, more prominent in the cortex than medulla (p < .05 for all). In contrast, mice that overexpressed CuZn SOD did not differ in the amount of apoptosis after CLP compared with genetically normal mice (p = NS for all)., Conclusions: Oxidative stress occurs in sepsis and appears to be one stimulus for the development of lymphocyte apoptosis, a process that is partly regulated by CuZn SOD. However, we were unable to demonstrate that overexpression of this enzyme suppressed lymphocyte apoptosis, suggesting that either other antioxidant defenses or other pathways independent of oxidative stress may mediate lymphocyte elimination in this syndrome.

Trisomy 21 (Down syndrome) is the most common chromosomal abnormality associated with mental retardation in humans. Sim2, a human homologue of Drosophila sim gene, which acts as a master regulator of the early development of the fly central nervous system midline, is located on chromosome 21, in the Down syndrome critical region, and might therefore be involved in the pathogenesis of some of the morphological features and brain anomalies observed in Down syndrome. We report here the detailed expression pattern of murine mSim2 gene in Ts1Cje mice fetuses, a segmental trisomy 16 mouse model for trisomy 21, and its overexpression in the zona limitans of the diencephalon using a new quantitative method based on the whole-mount RNA hybridization technique.

Aquaporin-3 (AQP3) is a water channel expressed at the basolateral plasma membrane of kidney collecting-duct epithelial cells. The mouse AQP3 cDNA was isolated and encodes a 292-amino acid water/glycerol-transporting glycoprotein expressed in kidney, large airways, eye, urinary bladder, skin, and gastrointestinal tract. The mouse AQP3 gene was analyzed, and AQP3 null mice were generated by targeted gene disruption. The growth and phenotype of AQP3 null mice were grossly normal except for polyuria. AQP3 deletion had little effect on AQP1 or AQP4 protein expression but decreased AQP2 protein expression particularly in renal cortex. Fluid consumption in AQP3 null mice was more than 10-fold greater than that in wild-type litter mates, and urine osmolality (<275 milliosmol) was much lower than in wild-type mice (>1,200 milliosmol). After 1-desamino-8-d-arginine-vasopressin administration or water deprivation, the AQP3 null mice were able to concentrate their urine partially to approximately 30% of that in wild-type mice. Osmotic water permeability of cortical collecting-duct basolateral membrane, measured by a spatial filtering optics method, was >3-fold reduced by AQP3 deletion. To test the hypothesis that the residual concentrating ability of AQP3 null mice was due to the inner medullary collecting-duct water channel AQP4, AQP3/AQP4 double-knockout mice were generated. The double-knockout mice had greater impairment of urinary-concentrating ability than did the AQP3 single-knockout mice. Our findings establish a form of nephrogenic diabetes insipidus produced by impaired water permeability in collecting-duct basolateral membrane. Basolateral membrane aquaporins may thus provide blood-accessible targets for drug discovery of aquaretic inhibitors.

Oxidative damage has been implicated in the aging process and in a number of degenerative diseases. To investigate the role of oxygen radicals in the aging process in mammals, the life spans of transgenic mice on a CD-1 background expressing increased levels of CuZn superoxide dismutase (CuZnSOD), the enzyme that metabolizes superoxide radicals, were determined. Homozygous transgenic mice with a two- to five-fold elevation of CuZnSOD in various tissues showed a slight reduction of life span, whereas hemizygous mice with a 15- to 3-fold increase of CuZnSOD showed no difference in life span from that of the nontransgenic littermate controls. The results suggest that constitutive and ubiquitous overexpression of CuZnSOD alone is not sufficient to extend the life spans of transgenic mice.

Long-term potentiation (LTP) and depression (LTD) were investigated in hippocampus of a genetic model of Down syndrome, the segmental trisomy (Ts65Dn) mouse. Field excitatory postsynaptic potentials were recorded from hippocampal slices and LTP and LTD evoked sequentially. LTP decreased whereas LTD increased significantly in Ts65Dn compared with control hippocampus.

Deta nonoate (deta-NO), a zwitterion nitric oxide (NO) donor, potently inhibited forskolin- and angiotensin II-stimulated aldosterone production in human adrenocortical H295R cells in a concentration-dependent manner (0.1-1000 microM). The half-maximal and maximal inhibition of forskolin-evoked aldosteronogenesis occurred at 0.6 and 100 microM deta-NO, respectively. The respective half-maximal and maximal deta-NO-mediated inhibition of angiotensin II-stimulated aldosterone generation occurred at 150 microM and 1 mM. In H295R cells, deta-NO and sodium nitroprusside did not stimulate cGMP production, and the soluble guanylyl cyclase inhibitor oxadiazoloquinoxalinone (10 microM) did not block deta-NO-mediated attenuation of aldosteronogenesis. 25-Hydroxycholesterol (10 microM)-facilitated aldosterone synthesis was also diminished with half-maximal and maximal inhibition occurring at 120 microM and 1 mM deta-NO, respectively. Taken together, these results demonstrate that NO inhibits human aldosteronogenesis without stimulating guanylyl cyclase in H295R cells.

Aquaporin-5 (AQP5) is a water-selective transporting protein expressed in epithelial cells of serous acini in salivary gland. We generated AQP5 null mice by targeted gene disruption. The genotype distribution from intercross of founder AQP5 heterozygous mice was 70:69:29 wild-type:heterozygote:knockout, indicating impaired prenatal survival of the null mice. The knockout mice had grossly normal appearance, but grew approximately 20% slower than litter-matched wild-type mice when placed on solid food after weaning. Pilocarpine-stimulated saliva production was reduced by more than 60% in AQP5 knockout mice. Compared with the saliva from wild-type mice, the saliva from knockout mice was hypertonic (420 mosM) and dramatically more viscous. Amylase and protein secretion, functions of salivary mucous cells, were not affected by AQP5 deletion. Water channels AQP1 and AQP4 have also been localized to salivary gland; however, pilocarpine stimulation studies showed no defect in the volume or composition of saliva in AQP1 and AQP4 knockout mice. These results implicate a key role for AQP5 in saliva fluid secretion and provide direct evidence that high epithelial cell membrane water permeability is required for active, near-isosmolar fluid transport.

The regulation of MUC2, a major goblet cell mucin gene, was examined by constructing transgenic mice containing bases -2864 to +17 of the human MUC2 5'-flanking region fused into the 5'-untranslated region of a human growth hormone (hGH) reporter gene. Four of eight transgenic lines expressed reporter. hGH message expression was highest in the distal small intestine, with only one line expressing comparable levels in the colon. This contrasts with endogenous MUC2 expression, which is expressed at its highest levels in the colon. Immunohistochemical analysis indicated that goblet cell-specific expression of reporter begins deep in the crypts, as does endogenous MUC2 gene expression. These results indicate that the MUC2 5'-flanking sequence contains elements sufficient for the appropriate expression of MUC2 in small intestinal goblet cells. Conversely, elements located outside this region appear necessary for efficient colonic expression, implying that the two tissues utilize different regulatory elements. Thus many, but not all, of the elements necessary for MUC2 gene regulation reside between bases -2864 and +17 of the 5'-flanking region.

To distinguish the role of Mn superoxide dismutase (MnSOD) from that of cytoplasmic CuZn superoxide dismutase (CuZnSOD), the mouse MnSOD gene (Sod2) was inactivated by homologous recombination. Sod2 -/- mice on a CD1 (outbred) genetic background die within the first 10 days of life (mean, 5.4 days) with a complex phenotype that includes dilated cardiomyopathy, accumulation of lipid in liver and skeletal muscle, metabolic acidosis and ketosis, and a severe reduction in succinate dehydrogenase (complex II) and aconitase (a TCA cycle enzyme) activities in the heart and, to a lesser extent, in other organs. These findings indicate that MnSOD is required to maintain the integrity of mitochondrial enzymes susceptible to direct inactivation by superoxide. On the other hand, Lebovitz et al. reported an independently derived MnSod null mouse (Sod2tmlLeb) on a mixed C57BL/6 and 129Sv background with a different phenotype. Because a difference in genetic background is the most likely explanation for the phenotypic differences, the two mutant lines were crossed into different genetic backgrounds for further analyses. To study the phenotype of Sod2tmlLeb mice CD1 background, the Sod2tmlLeb mice were crossed to CD1 for two generations before the -/+ mice were intercrossed to generate -/- mice. The life span distribution of CD1 < Sod2-/- > Leb was shifted to the left, indicating a shortened life span on the CD1 background. Furthermore, the CD1 < Sod2-/- > Leb mice develop metabolic acidosis at an early stage as was observed with CD1 < Sod2-/- > Cje. When Sod2tmlCje was placed on C57BL/6J (B6) background, the -/- mice were found to die either during midgestation or within the first 4 days after birth. However, when the B6 < Sod2 -/+ > Cje were crossed with DBA/2J (D2) for the generation of B6D2F2 < Sod2-/- > Cje mice, an entirely different phenotype, similar to that described by Lebovitz et al., was observed. The F2 Sod -/- mice were able to survive up to 18 days, and the animals that lived for more than 15 days displayed neurological abnormalities including ataxia and seizures. Their hearts were not as severely affected as were those of the CD1 mice, and neurological degeneration rather than heart defect appears to be the cause of death.

The localization of substance P in brain regions that coordinate stress responses and receive convergent monoaminergic innervation suggested that substance P antagonists might have psychotherapeutic properties. Like clinically used antidepressant and anxiolytic drugs, substance P antagonists suppressed isolation-induced vocalizations in guinea pigs. In a placebo-controlled trial in patients with moderate to severe major depression, robust antidepressant effects of the substance P antagonist MK-869 were consistently observed. In preclinical studies, substance P antagonists did not interact with monoamine systems in the manner seen with established antidepressant drugs. These findings suggest that substance P may play an important role in psychiatric disorders.

Oxidative injury underlies the cellular injury and cell death in a variety of disease states. In muscular dystrophies, evidence from in vivo and in vitro studies suggests that muscle degeneration may be secondary to an increased susceptibility to oxidative stress. To address the role of free radical metabolism in the pathogenetic process of muscular dystrophies, we examined the muscle of transgenic mice that overexpress copper/zinc (Cu/Zn) superoxide dismutase. Overexpression of this enzyme can sensitize cells to oxidative injury, and Cu/Zn superoxide dismutase activity was elevated approximately fourfold above control levels in skeletal muscle of the transgenic strain. Examination of serum creatine phosphokinase levels in these mice revealed significant elevations after 2 months of age, indicative of active muscle breakdown. By 8 months of age, there was gross atrophy of the quadriceps muscle, and other hindlimb muscles were variably affected. Histologically, there was evidence of widespread muscle necrosis and regeneration, fiber splitting, and replacement of muscle with adipose and fibrous connective tissue, typical of a muscular dystrophy. Associated with the development of this degeneration was an increase in the levels of lipid peroxidation in the muscle of Cu/Zn superoxide dismutase transgenic mice, highlighting the central role of oxidative injury in this pathogenetic process. These results demonstrate that oxidative damage can be the primary pathogenetic process underlying a muscular dystrophy.

A mouse model for Down syndrome, Ts1Cje, has been developed. This model has made possible a step in the genetic dissection of the learning, behavioral, and neurological abnormalities associated with segmental trisomy for the region of mouse chromosome 16 homologous with the so-called "Down syndrome region" of human chromosome segment 21q22. Tests of learning in the Morris water maze and assessment of spontaneous locomotor activity reveal distinct learning and behavioral abnormalities, some of which are indicative of hippocampal dysfunction. The triplicated region in Ts1Cje, from Sod1 to Mx1, is smaller than that in Ts65Dn, another segmental trisomy 16 mouse, and the learning deficits in Ts1Cje are less severe than those in Ts65Dn. In addition, degeneration of basal forebrain cholinergic neurons, which was observed in Ts65Dn, was absent in Ts1Cje.

The excitatory neurotransmitter glutamate coexists with the peptide known as substance P in primary afferents that respond to painful stimulation. Because blockers of glutamate receptors reliably reduce pain behaviour, it is assumed that 'pain' messages are mediated by glutamate action on dorsal horn neurons. The contribution of substance P, however, is still unclear. We have now disrupted the mouse preprotachykinin A gene (PPT-A), which encodes substance P and a related tachykinin, neurokinin A. We find that although the behavioural response to mildly painful stimuli is intact in these mice, the response to moderate to intense pain is significantly reduced. Neurogenic inflammation, which results from peripheral release of substance P and neurokinin A, is almost absent in the mutant mice. We conclude that the release of tachykinins from primary afferent pain-sensing receptors (nociceptors) is required to produce moderate to intense pain.