GAA trinucleotide repeat region regulates M9/pMGA gene expression in Mycoplasma gallisepticum

Mycoplasma gallisepticum, the cause of chronic respiratory infections in the avian host, possesses a family of M9/pMGA genes encoding an adhesin(s) associated with hemagglutination. Nucleotide sequences of M9/pMGA gene family members indicate extensive sequence similarity in the promoter regions of both the transcribed and silent genes. The mechanism that regulates M9/pMGA gene expression is unknown, but studies have revealed an apparent correlation between gene expression and the number of tandem GAA repeat motifs located upstream of the putative promoter. In this study, transposon Tn4001 was used as a vector with the Escherichia coli lacZ gene as the reporter system to examine the role of the GAA repeats in M9/pMGA gene expression in M. gallisepticum. A 336-bp M9 gene fragment (containing the GAA repeat region, the promoter, and the translation start codon) was amplified by PCR, ligated with a lacZ gene from E. coli, and inserted into the Tn4001-containing plasmid pISM2062. This construct was transformed into M. gallisepticum PG31. Transformants were filter cloned on agar supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to monitor lacZ gene expression on the basis of blue/white color selection. Several cycles of filter cloning resulted in cell lineages in which lacZ gene expression alternated between the On and Off states in successive generations of progeny clones. The promoter regions of the M9-lacZ hybrid genes of individual progeny clones were amplified by PCR and sequenced. The only differences between the promoter regions of the blue and white colonies were in the number of GAA repeats. Clones that expressed lacZ had exactly 12 tandem copies of the GAA repeat. Clones that did not express lacZ invariably had either more than 12 (14 to 16) or fewer than 12 (5 to 11) GAA repeats. Southern analysis of M. gallisepticum chromosomal DNA confirmed that the phase-variable expression of the lacZ reporter gene was not caused by Tn4001 transposition. These data strongly indicate that changes in the length of the GAA repeat region are responsible for regulating M9/pMGA gene expression.     

Polymorphic MHC ancestral haplotypes affect the activity of tumour necrosis factor-alpha.

It remains unclear which MHC loci are involved in susceptibility to autoimmune diseases and immune deficiencies. We have chosen to evaluate whether different alleles of tumour necrosis factor-alpha (TNF-alpha) are important, as TNF has been implicated in the etiology of many immunological disorders. We have shown previously that a restriction fragment length polymorphism in the TNF region correlates with MHC ancestral haplotypes. We therefore examined the effect of ancestral haplotype on the activity of TNF-alpha in culture supernatants of lymphoblastoid cell lines. The results demonstrate that TNF-alpha activity in supernatants of 8.1 (A1, B8, DR3) cell lines was higher than that present in the supernatants from cells homozygous for eight different MHC ancestral haplotypes, and indicate that polymorphisms in TNF-alpha, or in other MHC genes that regulate TNF, may be responsible for the 8.1 phenotype.     

Relationship between fatty liver and subsequent development of necrosis, inflammation and fibrosis in experimental alcoholic liver disease

Rats fed a diet varying in the amount of fat, infused with ethanol, were studied to determine the relationship among diet, degree of fatty liver, and development of necrosis, inflammation, and fibrosis. Three groups of experimental animals, male Wistar rats, were fed diets containing 25% fat, 35% fat, and 32% fat with low protein. Morphologic assessment of liver injury was performed monthly by obtaining liver biopsies. The greatest degree of fatty infiltration at 1 month was seen in the high fat-low protein group, the mean fat score (3.8 +/- 0.37) was significantly higher than in the other two groups (P less than 0.05 and P less than 0.01). When the subsequent development of necrosis, inflammation, and fibrosis was related to the degree of fatty infiltration at 1 month, a significant relationship was seen between the number of animals developing these pathologic lesions and the severity of fatty liver. Our results show that the degree of fatty infiltration of the liver, influenced by the dietary intake of both fat and protein, is related to the subsequent development of necrosis, inflammation, and fibrosis in our intragastric feeding model for alcoholic liver disease.     

CYP2E1 inhibition enhances mallory body formation

Mallory body (MB) formation is a complex phenomenon seen in chronic liver disease. CYP2E1 may play a role in preventing MB formation since it is involved in the elimination of toxic drugs and chemicals. When mice were fed with diethyl-1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) for 10 weeks, Mallory bodies (MBs) developed in the liver at the end of this period. When DDC feeding was combined with CMZ (an efficient in vivo CYP2E1 inhibitor), more MBs formed compared to DDC feeding alone. DDC was shown to be a suicide inhibitor of CYP2E1. The level of CYP2E1 protein in the liver was further reduced by the DDC and CMZ treatment when measured by Western blot. To test whether CYP2E1 reduced MB formation, CYP2E1 knockout mice and CYP2E1 overexpressed mice were fed with DDC or DDC and CMZ for 10 weeks. MB formation increased markedly in the liver of CYP2E1 knockout mice when fed with DDC only. CYP2E1 overexpressed mice showed an increase in MB formation when the mice were fed with the combination of DDC and CMZ where the amount of CYP2E1 was reduced to levels seen in wild type mice. It was concluded that CYP2E1 inhibits MB formation by increasing the rate of elimination of DDC and/or its toxic intermediates.     

Clinical experience with cytomegalovirus isolation using conventional cell cultures and early antigen detection in centrifugation-enhanced shell vial cultures.

A total of 1,915 clinical samples was inoculated by low-speed centrifugation into shell vials (Bartels Immunodiagnostics, Bellvue, Wash.) containing cover slip monolayers of MRC-5 fibroblasts. At 1 and 2 days postinoculation, one cover slip was stained by an indirect immunofluorescence technique using a monoclonal antibody (Biotech Research Laboratories for Dupont, Billerica, Mass.) to cytomegalovirus (CMV) early antigen (EA). Clinical samples were also inoculated into three MRC-5 or MRHF cell cultures which were observed for 30 days for the appearance of a cytopathic effect (CPE). Of 157 CMV-positive samples, 92 (59%) were identified by centrifugation-enhanced EA (CE-EA) and 131 (83%) produced a CPE. CE-EA was less sensitive than CPE for all types of samples, although 17% of CMV-positive samples were detected by CE-EA alone. Evaluation of the CMV status of patients with CE-EA-positive-CPE-negative samples indicated that these samples likely represented true CMV-positive results. The average elapsed time between culture inoculation and identification of CMV decreased as follows when both CE-EA and CPE, rather than CPE alone, were used: urines, 15 to 7 days; buffy coats, 18 to 9 days; lung samples, 13 to 8 days; throat samples, 18 to 7 days. Although CE-EA was less sensitive than 30-day cell culture, both CE-EA and CPE were identified as valuable in CMV detection, and neither could be discontinued without a decrease in the CMV isolation rate or an increase in the turnaround time.     

Discriminating adaptive attributions: Agreement between self-report and objective ratings

According to the reformulated learned helplessness model of depression, causal attributions are an important mediator of the effects on mood of positive and negative experiences. Adaptive attributions for negative events are assumed to be external, unstable, and specific. In the present study, subjects exposed to one of two attribution training procedures or a control condition made attributions for hypothetical events under neutral and adaptive instructional sets. Attributions were rated by subjects and coders blind to the purpose of the study. Results indicated that subjects' views of adaptive causal attributions were congruent with predictions from the learned helplessness model. The ratings of the objective coders indicated that subjects' attributions really did change in response to the adaptive instructions in the predicted direction. Implications of these results for the reformulated learned helplessness model and depression therapies that include an attribution retraining component are discussed.The authors would like to thank Dan Russell for his very helpful comments on earlier drafts of this paper.     

Clinical utility of PTSD,resilience, sleep,and blast as risk factors to predict poor neurobehavioral functioning following traumatic brain injury: A longitudinal study in U.S. military service members

Quality of Life Research - This study examined the clinical utility of post-traumatic stress disorder (PTSD), low resilience, poor sleep, and lifetime blast exposure as risk factors for predicting...