Comparing the effects of subchronic phencyclidine and medial prefrontal cortex dysfunction on cognitive tests relevant to schizophrenia.

RATIONALE: It is becoming increasingly clear that the development of treatments for cognitive symptoms of schizophrenia requires urgent attention, and that valid animal models of relevant impairments are required. With subchronic psychotomimetic agent phencyclidine (scPCP), a putative model of such impairment, the extent to which changes following scPCP do or do not resemble those following dysfunction of the prefrontal cortex is of importance. OBJECTIVES: The present study carried out a comparison of the most common scPCP dosing regimen with excitotoxin-induced medial prefrontal cortex (mPFC) dysfunction in rats, across several cognitive tests relevant to schizophrenia. METHODS: ScPCP subjects were dosed intraperitoneal with 5 mg/kg PCP or vehicle twice daily for 1 week followed by 1 week washout prior to behavioural testing. mPFC dysfunction was induced via fibre-sparing excitotoxin infused into the pre-limbic and infralimbic cortex. Subjects were tested on spontaneous novel object recognition, touchscreen object-location paired-associates learning and touchscreen reversal learning. RESULTS: A double-dissociation was observed between object-location paired-associates learning and object recognition: mPFC dysfunction impaired acquisition of the object-location task but not spontaneous novel object recognition, while scPCP impaired spontaneous novel object recognition but not object-location associative learning. Both scPCP and mPFC dysfunction resulted in a similar facilitation of reversal learning. CONCLUSIONS: The pattern of impairment following scPCP raises questions around its efficacy as a model of cognitive impairment in schizophrenia, particularly if importance is placed on faithfully replicating the effects of mPFC dysfunction.

In vivo interactions between ionizing radiation, inflammation and chemical carcinogens identified by increased DNA damage responses.

Exposure to ionizing radiation or a variety of chemical agents is known to increase the risk of developing malignancy and many tumors have been linked to inflammatory processes. In most studies, the potentially harmful effects of ionizing radiation or other agents are considered in isolation, mainly due to the large number of experiments required to assess the effects of mixed exposures with different doses and different schedules, and the length of time and expense of studies using disease as the measure of outcome. Here, we have used short-term DNA damage responses to identify interactive effects of mixed exposures. The data demonstrate that exposure to ionizing radiation on two separate occasions ten days apart leads to an increase in the percentage of cells with a sub-G(0) DNA content compared to cells exposed only once, and this is a greater than additive effect. Short-term measurements of p53 stabilization, induction of p21/Cdkn1a and of apoptosis also identify these interactive effects. We also demonstrate similar interactive effects of radiation with the mutagenic chemical methyl-nitrosourea and with a nonspecific pro-inflammatory agent, lipopolysaccharide. The magnitude of the interactive effects is greater in cells taken from mice first exposed as juveniles compared to adults. These data indicate that short-term measurements of DNA damage and response to damage are useful for the identification of interactions between ionizing radiation and other agents.

New translational assays for preclinical modelling of cognition in schizophrenia: the touchscreen testing method for mice and rats.

We describe a touchscreen method that satisfies a proposed 'wish-list' of desirables for a cognitive testing method for assessing rodent models of schizophrenia. A number of tests relevant to schizophrenia research are described which are currently being developed and validated using this method. These tests can be used to study reward learning, memory, perceptual discrimination, object-place associative learning, attention, impulsivity, compulsivity, extinction, simple Pavlovian conditioning, and other constructs. The tests can be deployed using a 'flexible battery' approach to establish a cognitive profile for a particular mouse or rat model. We have found these tests to be capable of detecting not just impairments in function, but enhancements as well, which is essential for testing putative cognitive therapies. New tests are being continuously developed, many of which may prove particularly valuable for schizophrenia research.

Readability and content of postoperative tonsillectomy instructions given to patients in Scotland, a completed audit cycle.

The aim of this study was to assess the impact of the recommendations and interventions of the 2003 audit of 'readability and content of postoperative tonsillectomy instructions, given to patients in Scotland'. A two-cycle audit of readability and content of postoperative tonsillectomy instructions was undertaken. All National Health Service (NHS) hospital wards and associated Otolaryngology Departments in Scotland where tonsillectomies were being performed were contacted. Interventions following the first cycle included the mailing copies of original audit results and conclusions to all ear, nose and throat wards in Scotland, presentation at a National Meeting and publication of results in a peer reviewed journal. While changes had occurred in 61% of the information sheets, and with six of the 31 (19%) postoperative information sheets now being written at or below the recommended reading level, the average reading grade/age required to understand these information sheets still remains above those recommended by patient education experts. In conclusion, the majority of postoperative tonsillectomy information sheets in Scotland remain written at a level above those recommended by patient education experts. The interventions undertaken in this audit were of limited success. The ENT-UK Tonsillectomy Information sheets (2006) are written at reasonable reading levels, have good content levels and we continue to recommend these information sheets.

Diagnosis of tuberculosis in the head and neck.

OBJECTIVES: To establish the features of Mycobacterium tuberculosis infection in the head and neck region, and to determine which investigations have the greatest diagnostic accuracy. STUDY DESIGN: Region-based, retrospective cohort study. METHOD: The study included 148 patients with tuberculosis of the head and neck treated in the Greater Glasgow and Clyde region between 2000 and 2007. RESULTS: The following diagnostic sensitivities were calculated: 53 per cent for fine needle aspiration, 95 per cent for core biopsy and 91 per cent for lymph node excision biopsy. There was a statistically significant difference between the sensitivity results for fine needle aspiration versus core biopsy (p = 0.0003) and fine needle aspiration versus excision biopsy (p < 0.0001). There was no statistically significant difference between the sensitivity results for core biopsy and excision biopsy. CONCLUSION: Core biopsy has equivalent diagnostic accuracy to excision biopsy in the investigation of head and neck tuberculosis. We suggest that core biopsy should be used in preference to lymph node excision, as it can be performed under local anaesthetic outside the operating theatre. A proposed algorithm for diagnostic management is included.

Neck lumps and head and neck tumours in children.

This article discusses the presentation, investigation and treatment of both benign and malignant lumps encountered in the head and neck region in children.

Mammary tumor induction and premature ovarian failure in ApcMin mice are not enhanced by Brca2 deficiency.

Inherited BRCA2 mutations predispose individuals to breast cancer and increase risk at other sites. Recent studies have suggested a role for the APC I1307K allele as a low-penetrance breast cancer susceptibility gene that enhances the phenotypic effects of BRCA1 and BRCA2 mutations. To model the consequences of inheriting mutant alleles of the BRCA2 and APC tumor suppressor genes, we examined tumor outcome in C57BL/6 mice with mutations in the Brca2 and Apc genes. We hypothesized that if the Brca2 and Apc genes were interacting to influence mammary tumor susceptibility, then mammary tumor incidence and/or multiplicity would be altered in mice that had inherited mutations in both genes. Female and male offspring treated with a single IP injection of 50 mg/kg N-ethyl-N-nitrosourea (ENU) at 35 days of age developed mammary adenoacanthomas by 100 days of age. The female Apc-mutant and Brca2/Apc double-mutant progeny had mean mammary tumor multiplicities of 6.7+/-2.8 and 7.2+/-2.7, respectively, compared to wild-type and Brca2-mutant females, which had mean mammary tumor multiplicities of 0.1+/-0.4 and 0.3+/-0.5, respectively. Female ENU-treated Apc-mutant and Brca2/Apc double heterozygotes were also susceptible to premature ovarian failure. Thus, the inheritance of an Apc mutation predisposes ENU-treated female and male mice to mammary tumors and, in the case of female mice, to ovarian failure. These results indicate that mammary tumor development in Apc-mutant mice can progress independently of ovarian hormones. The Apc mutation-driven phenotypes were not modified by mutation of Brca2, perhaps because Brca2 acts in a hormonally dependent pathway of mammary carcinogenesis.

Crystal structure of Streptococcus pneumoniae acyl carrier protein synthase: an essential enzyme in bacterial fatty acid biosynthesis.

Acyl carrier protein synthase (AcpS) catalyzes the formation of holo-ACP, which mediates the essential transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and lipids in the cell. Thus, AcpS plays an important role in bacterial fatty acid and lipid biosynthesis, making it an attractive target for therapeutic intervention. We have determined, for the first time, the crystal structure of the Streptococcus pneumoniae AcpS and AcpS complexed with 3'5'-ADP, a product of AcpS, at 2.0 and 1.9 A resolution, respectively. The crystal structure reveals an alpha/beta fold and shows that AcpS assembles as a tightly packed functional trimer, with a non-crystallographic pseudo-symmetric 3-fold axis, which contains three active sites at the interface between protomers. Only two active sites are occupied by the ligand molecules. Although there is virtually no sequence similarity between the S.pneumoniae AcpS and the Bacillus subtilis Sfp transferase, a striking structural similarity between both enzymes was observed. These data provide a starting point for structure-based drug design efforts towards the identification of AcpS inhibitors with potent antibacterial activity.

The role of tryptophan residues in substrate binding to catalytic domains A and B of xylanase C from Fibrobacter succinogenes S85.

Oxidation of the isolated catalytic domain B of xylanase C (XynC-B) from Fibrobacter succinogenes with N-bromosuccinimide (NBS) resulted in the modification of five of the seven Trp residues present in the enzyme. Hydrolytic activity of the enzyme was rapidly lost upon initiation of oxidation as a molar ratio of about two NBS molecules per molar equivalent of protein was sufficient to cause 50% inhibition of enzyme activity, and the addition of five molar equivalents of NBS resulted in less than 10% activity. Pre-incubation of XynC-B with the competitive inhibitor D-xylose resulted in the apparent protection of two Trp residues from oxidation. Xylose protection of the enzyme also resulted in a maintenance of activity, with 60% activity still evident after addition of 8-9 molar equivalents of NBS. This protection from inactivation was enhanced by the inclusion of xylohexaose in reaction mixtures. Under these conditions, however, a further Trp residue was protected from NBS oxidation. The three protected Trp residues were identified as Trp135, Trp161 and Trp202 by differential labelling and peptide mapping of NBS-oxidized preparations of the xylanase employing a combination of electrospray mass spectroscopic analysis and N-terminal sequencing. By analogy to the known structures of the family 11 xylanases, the fully conserved Trp202 residue is located on the only alpha-helix present in the enzymes, at the interface between it and the back of the beta-sheet which forms the active site cleft. Trp135 represents a highly conserved aromatic residue in family 11, but it is replaced with Thr in domain A of F. succinogenes xylanase C. To investigate the role of Trp135 in conferring the different activity profile of domain B relative to domain A, the Trp135Thr and Trp135Ala derivatives of domain B were prepared by site-directed mutagenesis. However, the kinetic parameters of the two domain B derivatives were not significantly different compared to the wild-type enzyme as reflected by K(M) and k(cat) values and product distribution profiles. Similar results were obtained with the Trp161Ala derivative of domain B, indicating that these two residues do not directly participate in the binding of substrate but likely form the foundation for binding subsite 2.

Characterization of function and activity of domains A, B and C of xylanase C from Fibrobacter succinogenes S85.

Xylanase C from the ruminant bacterium Fibrobacter succinogenes is comprised of two catalytic domains, A and B, and a third domain, C, of unknown function. The DNA coding for domains A and B of xylanase C were separately cloned and expressed in Escherichia coli as fusion proteins with glutathione-S:-transferase. The fusion proteins were isolated by affinity chromatography on glutathione-Sepharose 4B, cleaved with thrombin and the released xylanase C catalytic domains A and B were purified to apparent homogeneity by anion-exchange chromatography on Mono Q. Electrospray mass spectrometry provided a molecular mass of 27 818 Da (expected, 27 820 Da) for domain B. The pH and temperature optima for activity of domain B on oat spelt xylan were 5.0 and 52 degrees C, respectively. A kinetic analysis of the activity of the catalytic domain A on oat spelt xylan, birch wood xylan and xylooligomers at pH 6.5 and 37 degrees C provided data significantly different to those obtained previously with a protease-derived form of the enzyme [Zhu et al. (1994) J. Bacteriol. 176, 3885-3894]. The isolated domain A was more active on barley-glucan than the protease-derived form and its affinity for birch wood xylan was enhanced resulting in greater overall catalytic efficiency as reflected by k(cat)/K:(M) values. Likewise, significant differences in the Michaelis-Menten parameters K:(M), k(cat) and k(cat)/K:(M) were obtained with domain B compared with values previously reported with this domain attached to domain C. In general, the presence of domain C appeared to decrease the overall efficiency of domain B 7- and 36-fold with birch wood xylan and xylopentaose as substrates, respectively, as reflected by values of k(cat)/K:(M). The removal of domain C also affected the mode of action of domain B such that it more closely resembled that of catalytic domain A. However, no change in either pH and temperature optima or stability were found with domain B compared with the combined domains B and C. The function of domain C remains unknown, but hydrophobic cluster analysis indicated that it may belong to a class of dockerin domains involved in the protein-protein interactions of cellulolytic and xylanolytic complexes.

BRCA2-null embryonic survival is prolonged on the BALB/c genetic background.

Women who inherit mutations in the BRCA2 cancer susceptibility gene have an 85% chance of developing breast cancer. The function of the BRCA2 gene remains elusive, but there is evidence to support its role in transcriptional transactivation, tumor suppression, and the maintenance of genomic integrity. Individuals with identical BRCA2 mutations display a different distribution of cancers, suggesting that there are low-penetrance genes that can modify disease outcome. We hypothesized that genetic background could influence embryonic survival of a Brca2 mutation in mice. Brca2-null embryos with a 129/SvEv genetic background (129(B2-/-)) died before embryonic day 8. 5. Transfer of this Brca2 mutation onto the BALB/cJ genetic background (BALB/c(B2-/-)) extended survival to embryonic day 10.5. These results indicate that the BALB/c background harbors genetic modifiers that can prolong Brca2-null embryonic survival. The extended survival of BALB/c(B2-/-) embryos enabled us to ask whether transcriptional regulation of the Brca1 and Brca2 genes is interdependent. The interdependence of Brca1 and Brca2 was evaluated by studying Brca2 gene expression in BALB/c(B1-/-) embryos and Brca1 gene expression in BALB/c(B2-/-) embryos. Nonisotopic in situ hybridization demonstrated that Brca2 transcript levels were comparable in BALB/c(B1-/-) embryos and wild-type littermates. Likewise, reverse transcriptase-polymerase chain reactions confirmed Brca1 mRNA expression in embryonic day 8.5 BALB/c(B2-/-) embryos that was comparable to Brca2-heterozygous littermates. Thus, the Brca1 and Brca2 transcripts are expressed independently of one another in Brca1- and Brca2-null embryos. Mol. Carcinog. 28:174-183, 2000.

Mice heterozygous for a Brca1 or Brca2 mutation display distinct mammary gland and ovarian phenotypes in response to diethylstilbestrol.

Women who inherit mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, are predisposed to the development of breast and ovarian cancer. We used mice with a Brca1 mutation on a BALB/cJ inbred background (BALB/cB1+/- mice) or a Brca2 genetic alteration on the 129/SvEv genetic background (129B2+/- mice) to investigate potential gene-environment interactions between defects in these genes and treatment with the highly estrogenic compound diethylstilbestrol (DES). Beginning at 3 weeks of age, BALB/cB1+/-, 129B2+/-, and wild-type female mice were fed a control diet or a diet containing 640 ppb DES for 26 weeks. DES treatment caused vaginal epithelial hyperplasia and hyperkeratosis, uterine inflammation, adenomyosis, and fibrosis, as well as oviductal smooth muscle hypertrophy. The severity of the DES response was mouse strain specific. The estrogen-responsive 129/SvEv strain exhibited an extreme response in the reproductive tract, whereas the effect in BALB/cJ and C3H/HeN(MMTV-) mice was less severe. The Brca1 and Brca2 genetic alterations influenced the phenotypic response of BALB/cJ and 129/SvEv inbred strains, respectively, to DES in the mammary gland and ovary. The mammary duct branching morphology was inhibited in DES-treated BALB/cB1+/- mice compared with similarly treated BALB/cB1+/+ littermates. In addition, the majority of BALB/cB1+/- mice had atrophied ovaries, whereas wild-type littermates were largely diagnosed with arrested follicular development. The mammary ductal architecture in untreated 129B2+/- mice revealed a subtle inhibited branching phenotype that was enhanced with DES treatment. However, no significant differences were observed in ovarian pathology between 129B2+/+ and 129B2+/- mice. These data suggest that estrogenic compounds may modulate mammary gland or ovarian morphology in BALB/cB1+/- and 129B2+/- mice. These observations are consistent with the hypothesis that compromised DNA repair processes in cells harboring Brca1 or Brca2 mutations lead to inhibited growth and differentiation compared with the proliferative response of wild-type cells to DES treatment.

Biochemical and molecular analyses of the Streptococcus pneumoniae acyl carrier protein synthase, an enzyme essential for fatty acid biosynthesis.

Acyl carrier protein synthase (AcpS) is an essential enzyme in the biosynthesis of fatty acids in all bacteria. AcpS catalyzes the transfer of 4'-phosphopantetheine from coenzyme A (CoA) to apo-ACP, thus converting apo-ACP to holo-ACP that serves as an acyl carrier for the biosynthesis of fatty acids and lipids. To further understand the physiological role of AcpS, we identified, cloned, and expressed the acpS and acpP genes of Streptococcus pneumoniae and purified both products to homogeneity. Both acpS and acpP form operons with the genes whose functions are required for other cellular metabolism. The acpS gene complements an Escherichia coli mutant defective in the production of AcpS and appears to be essential for the growth of S. pneumoniae. Gel filtration and cross-linking analyses establish that purified AcpS exists as a homotrimer. AcpS activity was significantly stimulated by apo-ACP at concentrations over 10 microm and slightly inhibited at concentrations of 5-10 microm. Double reciprocal analysis of initial velocities of AcpS at various concentrations of CoA or apo-ACP indicated a random or compulsory ordered bi bi type of reaction mechanism. Further analysis of the inhibition kinetics of the product (3',5'-ADP) suggested that it is competitive with respect to CoA but mixed (competitive and noncompetitive) with respect to apo-ACP. Finally, apo-ACP bound tightly to AcpS in the absence of CoA, but CoA failed to do so in the absence of apo-ACP. Together, these results suggest that AcpS may be allosterically regulated by apo-ACP and probably proceeds by an ordered reaction mechanism with the first formation of the AcpS-apo-ACP complex and the subsequent transfer of 4'-phosphopantetheine to the apo-ACP of the complex.

Identification and characterization of the penicillin-binding protein 2a of Streptococcus pneumoniae and its possible role in resistance to beta-lactam antibiotics.

To further understand the role of penicillin-binding protein 2a (PBP 2a) of Streptococcus pneumoniae in penicillin resistance, we confirmed the identity of the protein as PBP 2a. The PBP 2a protein migrated electrophoretically to a position corresponding to that of PBP 2x, PBP 2a, and PBP 2b of S. pneumoniae and was absent in a pbp2a insertional mutant of S. pneumoniae. We found that the affinities of PBP 2a for penicillins were lower than for cephalosporins and a carbapenem. When compared with other S. pneumoniae PBPs, PBP 2a exhibited lower affinities for beta-lactam antibiotics, especially penicillins. Therefore, PBP 2a is a low-affinity PBP for beta-lactam antibiotics in S. pneumoniae.

Extragonadal teratocarcinoma in chimeric mice.

Chimeric mice often are created through the genetic manipulation of the mouse embryo in the process of developing animal models of disease. These mice have variable percentages of their somatic and germ cells derived from the donor embryonic stem cells and host blastocysts. In the development of mouse models deficient in the breast cancer susceptibility gene 2 (Brca2) or the 70-kd heat shock protein (Hsp70-2), 3-4-week-old chimeras developed single or multiple masses composed of both well-differentiated and poorly differentiated tissues derived from all three germ layers. These cases of extragonadal teratocarcinoma, a rarely reported tumor, may be related to the genetic predisposition of the 129/Ola mouse strain used to generate the embryonic stem cells.

Mutation and expression analysis of the endoglin gene in hereditary hemorrhagic telangiectasia reveals null alleles.

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disorder characterized by multisystemic vascular dysplasia and recurrent hemorrhage from the sites of vascular lesions. Two genes have been identified for HHT. Endoglin, a TGF-beta binding protein which maps to chromosome 9q3, is the gene for HHT1. The type and location of most of the previously described mutations in the endoglin (ENG) gene suggested a dominant-negative model of receptor-complex dysfunction for the molecular basis of this disorder. In this article we describe 11 novel ENG mutations in HHT kindreds, which include missense and splice-site mutations. Two identical missense mutations in unrelated families disrupt the start codon of the gene. In addition, some frameshift and nonsense mutations lead to very low or undetectable levels of transcript from the mutant allele. These combined data suggest that the nature of most ENG mutations is to create a null (nonfunctional) allele, and that there is no requirement for the synthesis of a truncated endoglin protein in the pathogenesis of HHT.

Brca1 and Brca2 expression patterns in mitotic and meiotic cells of mice.

The mouse homologues of the breast cancer susceptibility genes, Brca1 and Brca2, are expressed in a cell cycle-dependent fashion in vitro and appear to be regulated by similar or overlapping pathways. Therefore, we compared the non isotopic in situ hybridization expression patterns of Brca1 and Brca2 mRNA in vivo in mitotic and meiotic cells during mouse embryogenesis, mammary gland development, and in adult tissues including testes, ovaries, and hormonally altered ovaries. Brca1 and Brca2 are expressed concordantly in proliferating cells of embryos, and the mammary gland undergoing morphogenesis and in most adult tissues. The expression pattern of Brca1 and Brca2 correlates with the localization of proliferating cell nuclear antigen, an indicator of proliferative activity. In the ovary, Brca1 and Brca2 exhibited a comparable hormone-independent pattern of expression in oocytes, granulosa cells and thecal cells of developing follicles. In the testes, Brca1 and Brca2 were expressed in mitotic spermatogonia and early meiotic prophase spermatocytes. Northern analyses of prepubertal mouse testes revealed that the time course of Brca2 expression was delayed in spermatogonia relative to Brca1. Thus, while Brca1 and Brca2 share concordant cell-specific patterns of expression in most proliferating tissues, these observations suggest that they may have distinct roles during meiosis.

Characterization of the rat and mouse homologues of the BRCA2 breast cancer susceptibility gene.

Inherited BRCA2 mutations confer profound susceptibility to human breast and ovarian cancer. The rat and mouse Brca2 homologues share 58% and 59% identity (72% similarity), respectively, with the human BRCA2 protein. The Brca2 proteins also share a potential nuclear localization signal (human codons 3263-3269) and a highly conserved large carboxyl region (77% identity, 86% similarity between human and rodents) that may represent important functional domains. At least six of eight previously described BRC repeats have been highly conserved in rats and mice. Expression studies demonstrate an 11-12 Kb transcript with rodent tissue-specific patterns of expression consistent with human BRCA2. These results will facilitate studies of Brca2 function during normal and neoplastic development.