Androgen receptor YAC transgenic mice carrying CAG 45 alleles show trinucleotide repeat instability

X-linked spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene. Disease-associated alleles (37-66 CAGs) change in length when transmitted from parents to offspring, with a significantly greater tendency to shift size when inherited paternally. As transgenic mice carrying human AR cDNAs with 45 and 66 CAG repeats do not display repeat instability, we attempted to model trinucleotide repeat instability by generating transgenic mice with yeast artificial chromosomes (YACs) carrying AR CAG repeat expansions in their genomic context. Studies of independent lines of AR YAC transgenic mice with CAG 45 alleles reveal intergenerational instability at an overall rate of approximately 10%. We also find that the 45 CAG repeat tracts are significantly more unstable with maternal transmission and as the transmitting mother ages. Of all the CAG/CTG repeat transgenic mice produced to date the AR YAC CAG 45 mice are unstable with the smallest trinucleotide repeat mutations, suggesting that the length threshold for repeat instability in the mouse may be lowered by including the appropriate flanking human DNA sequences. By sequence-tagged site content analysis and long range mapping we determined that one unstable transgenic line has integrated an approximately 70 kb segment of the AR locus due to fragmentation of the AR YAC. Identification of the cis - acting elements that permit CAG tract instability and the trans -acting factors that modulate repeat instability in the AR YAC CAG 45 mice may provide insights into the molecular basis of trinucleotide repeat instability in humans.      

RHD maternal-fetal genotype incompatibility and schizophrenia: extending the MFG test to include multiple siblings and birth order

Rh incompatibility disease (ie Rh hemolytic disease of the fetus and newborn) has been implicated as a risk factor for schizophrenia. Here, we extend the maternal-fetal genotype incompatibility (MFG) test used in an earlier case-parent trio study that found significant evidence for an increased risk of schizophrenia in RHD MFG-incompatible children. We modify the MFG test for case-parent trios to include any number of siblings. This modified test enables us to use more of the available data from the earlier study. The increased sample size not only gives us greater power to test for MFG incompatibility but it also enables us to model the impact of previous RHD MFG-incompatible pregnancies on the relative risk of RHD MFG incompatibility in later-born siblings. This modeling is important, because RHD MFG incompatibility is a proxy for Rh incompatibility disease, and the risk of Rh incompatibility disease increases with the number of previous RHD MFG-incompatible pregnancies. The best-fitting models are consistent with the hypothesized effect that previous incompatible pregnancies increase the risk of schizophrenia due to RHD MFG incompatibility. There was significant evidence that the relative risk of schizophrenia in the second- and later-born RHD MFG-incompatible children is 1.7, consistent with earlier estimates. Our extension of the MFG test has general application to family-based studies of maternal-genotype and MFG interaction effects.     

Comparative genomic indexing reveals the phylogenomics of Escherichia coli pathogens

The Escherichia coli O26 serogroup includes important food-borne pathogens associated with human and animal diarrheal disease. Current typing methods have revealed great genetic heterogeneity within the O26 group; the data are often inconsistent and focus only on verotoxin (VT)-positive O26 isolates. To improve current understanding of diversity within this serogroup, the genomic relatedness of VT-positive and -negative O26 strains was assessed by comparative genomic indexing. Our results clearly demonstrate that irrespective of virulence characteristics and pathotype designation, the O26 strains show greater genomic similarity to each other than to any other strain included in this study. Our data suggest that enteropathogenic and VT-expressing E. coli O26 strains represent the same clonal lineage and that VT-expressing E. coli O26 strains have gained additional virulence characteristics. Using this approach, we established the core genes which are central to the E. coli species and identified regions of variation from the E. coli K-12 chromosomal backbone.     

Macrophage antiviral activity: extrinsic versus intrinsic activity.

Peritoneal exudate cells from strains of mice both resistant and susceptible to challenge with mouse hepatitis virus strain JHM were examined for extrinsic and intrinsic antiviral activity. Thioglycolate-elicited and resident peritoneal cells from uninfected mice were able to suppress viral growth in a permissive cell. The active cell in both populations is an adherent, radiation-resistant, Thy-1.2 antigen- and Ia antigen-negative cell. The suppression of virus replication was not related to nonspecific cellular cytotoxicity directed against the permissive host cell, and no interferon was detected. The expression of extrinsic antiviral activity was not related to the ability of the host to resist mouse hepatitis virus infection by virtue of either age or genetic background. The expression of intrinsic antiviral activity, on the other hand, correlated with the ability of the host to resist virus challenge, indicating a characteristic distinction between these two in vitro mechanisms of macrophage-mediated antiviral activity with regard to host resistance to viral infection. Further, the ability of a macrophage to support viral replication itself was independent of the ability of the macrophage to suppress virus growth in another cell.     

Comparative growth dynamics and morphology between cultured myofibroblasts from granulating wounds and dermal fibroblasts

Rat myofibroblasts from granulating wound biopsies (RGW) were successfully cultured and compared in terms of growth and morphology with fibroblasts from uninjured rat dermis (RD). Populations of early passage (P-3) RGW myofibroblasts grew significantly more slowly than RD fibroblasts. Logarithmic growth was nearly the same in late passage (P-30) populations of both cell types. Early passage RGW myofibroblasts were similar to those in vivo, as shown by well-defined microfilament bundles. RD fibroblasts contained less well defined microfilaments. Both late passage RGW myofibroblasts and RD fibroblasts displayed evidence of morphologic dedifferentiation. These data show that morphologic features of myofibroblasts and fibroblasts in vivo are maintained in vitro. Evidence is presented that cultured animal myofibroblasts maintain differentiation in early passage, whereas late passage cells suggest that these differences disappear with time.     

Contrasting effects on HIF-1alpha regulation by disease-causing pVHL mutations correlate with patterns of tumourigenesis in von Hippel-Lindau disease

The von Hippel-Lindau tumour suppressor gene product (pVHL) associates with the elongin B and C and Cul2 proteins to form a ubiquitin-ligase complex (VCBC). To date, the only VCBC substrates identified are the hypoxia-inducible factor alpha subunits (HIF-1alpha and HIF-2alpha). However, pVHL is thought to have multiple functions and the significance of HIF-1alpha and HIF-2alpha regulation for tumour suppressor activity has not been defined. VHL disease is characterized by distinct clinical subtypes. Thus haemangioblastomas (HABs) and renal cell carcinoma (RCC) but not phaeochromocytoma (PHE) occur in type 1 VHL disease. Type 2 subtypes are characterized by PHE susceptibility but differ with respect to additional tumours (type 2A, PHE+HAB but not RCC; type 2B, PHE+ HAB+RCC; type 2C, PHE only). We investigated in detail the effect of 13 naturally occurring VHL mutations (11 missense), representing each phenotypic subclass, on HIF-alpha subunit regulation. Consistent effects on pVHL function were observed for all mutations within each subclass. Mutations associated with the PHE-only phenotype (type 2C) promoted HIF-alpha ubiquitylation in vitro and demonstrated wild-type binding patterns with pVHL interacting proteins, suggesting that loss of other pVHL functions are necessary for PHE susceptibility. Mutations causing HAB susceptibility (types 1, 2A and 2B) demonstrated variable effects on HIF-alpha subunit and elongin binding, but all resulted in defective HIF-alpha regulation and loss of p220 (fibronectin) binding. All RCC-associated mutations caused complete HIF-alpha dysregulation and loss of p220 (fibronectin) binding. Our findings are consistent with impaired ability to degrade HIF-alpha subunit being required for HAB development and RCC susceptibility.     

Histamine H1- and H2-receptor involvement in eosinophil infiltration and the microvascular changes associated with cutanneous anaphylaxis

Several substances alter eosinophil motility, but the relative importance of these putative mediators in immediate hypersensitivity remains unclear. The present study has reinvestigated the role of histamine in type I allergic eosinophil infiltration, and the temporally associated microvascular events, by examining the effect of H₁-and H₂-receptor antagonist pretreatment. A combination of cimetidine and pyrilamine significantly reduced eosinophil accumulation, whereas neither antagonist alone was effective. Similarly, cutaneous hyperemia, measured indirectly as ear surface temperature, was reduced only by the cimetidine-pyrilamine combination. Pyrilamine partially attenuated the increase in microvascular permeability, but the addition of cimetidine provided no further reduction.It appears that histamine participates significantly in mediating both the microvascular changes and the eosinophil infiltration evoked by cutaneous anaphylaxis. The histaminergic component of increased microvascular permeability appears to be an H₁-receptor mediated phenomenon. However, blockade of both H₁-and H₂-receptor subtypes is required to inhibit the hyperemia and eosinophil infiltration responses.     

Experimental gram-negative bacterial sepsis: prevention of mortality not preventable by antibiotics alone.

Outbred Swiss mice were inoculated intraperitoneally or intravenously with one 90 to 100% lethal dose of Escherichia coli O:18, Proteus mirabilis, or Klebsiella pneumoniae. After carefully timed intervals, aminoglycoside antibiotics were begun at dosages nnd intervals predetermined to constitute optimal therapy. With progressive increases in delay of antibiotic therapy, mortality rates increased progressively from 0% to 90 to 100%. Standardized models of infection were developed by selecting delay periods before initiating antibiotic therapy such that 50 to 70% mortalities resulted. Utilizing these models, agents with reputed anti-endotoxin activity were administered concomitantly with the delayed antibiotic therapy to determine if any could prevent gram-negative septic mortality no longer preventable by the antibiotics alone. The following were observed: (i) adrenal corticosteroids prevented mortality that was no longer preventable by optimal aminoglycoside antibiotics alone. The following were preventable by optimal aminoglycoside antibiotic therapy alone; (ii) specific antisera also did so, provided anaphylaxis was circumvented; (iii) in one model (P. mirabilis), such protection by adrenal corticosteroids and specific antiserum could be additive; (iv) adrenal corticosteroids and specific antiserum acted synergistically with the aminoglycoside antibiotics--no protection was achieved by delayed administration of the steroids or antiserum alone; (v) timing was crucial--the synergistic protective activity of adrenal corticosteroids and of specific antiserum with aminoglycosides declined rapidly as infection progressed; (vi) cyclophosphamide pretreatment markedly impaired the synergistic protective activity of specific antiserum and of adrenal corticosteroids with aminoglycosides; (vii) no reputed anti-endotoxin agents other than adrenal corticosteroids and specific antiserum proved capable of preventing mortality not preventable by aminoglycoside antibiotics alone. These included antisera to rough mutant Enterobacteriaceae of Rc, Rd, and Re chemotypes, anticoagulants (heparin), ascorbic acid, antiproteolytic agents (aprotinin), alpha adrenergic blockers (phenoxybenzamine), prostaglandin synthetase inhibitors (acetylsalicylic acid, sodium salicylate, indomethacin), nicotinamide, glucose, and insulin-glucose-potassium mixtures.