Monkeypox virus detection in rodents using real-time 3'-minor groove binder TaqMan assays on the Roche LightCycler

During the summer of 2003, an outbreak of human monkeypox occurred in the Midwest region of the United States. In all, 52 rodents suspected of being infected with monkeypox virus were collected from an exotic pet dealer and from private homes. The rodents were euthanized and submitted for testing to the United States Army Medical Research Institute of Infectious Diseases by the Galesburg Animal Disease Laboratory, Illinois Department of Agriculture. The rodent tissue samples were appropriately processed and then tested by using an integrated approach involving real-time polymerase chain reaction (PCR) assays, an antigen-detection immunoassay, and virus culture. We designed and extensively tested two specific real-time PCR assays for rapidly detecting monkeypox virus DNA using the Vaccinia virus F3L and N3R genes as targets. The assays were validated against panels of orthopox viral and miscellaneous bacterial DNAs. A pan-orthopox electrochemiluminescence (ECL) assay was used to further confirm the presence of Orthopoxvirus infection of the rodents. Seven of 12 (58%) animals (seven of 52 (15%) of all animals) tested positive in both monkeypox-specific PCR assays and two additional pan-orthopox PCR assays (in at least one tissue). The ECL results showed varying degrees of agreement with PCR. One hamster and three gerbils were positive by both PCR and ECL for all tissues tested. In addition, we attempted to verify the presence of monkeypox virus by culture on multiple cell lines, by immunohistology, and by electron microscopy, with negative results. Sequencing the PCR products from the samples indicated 100% identity with monkeypox virus strain Zaire-96-I-16 (a human isolate from the Congo). These real-time PCR and ECL assays represent a significant addition to the battery of tests for the detection of various orthopoxviruses. In light of the recent monkeypox virus transmissions, early detection of the virus is crucial for both natural outbreaks and potential acts of bioterrorism.     

Functional evidence for a breast cancer growth suppressor gene on chromosome 17

Rearrangements or deletions of chromosome 17 are the most frequentlyobserved genetic changes identified in breast tumors. Molecularanalyses suggest that in addition to the p53 gene on 17p13.1there may be at least three other tumor suppressor genes onchromosome 17 involved in breast cancer. Regions of loss ofheterozygosity (LOH) identified on 17p13.3 and 17q12-qter occurfrequently in breast tumors, and the BRCA-1 gene has been mappedto 17q21 by genetic linkage analysis. Here we provide biologicalevidence for the presence of a growth suppressor gene(s) onchromosome 17 that results In the In vitro growth suppressionof the p53 wild-type MCF 7 breast cancer cell line. We haveIntroduced a normal chromosome 17 into MCF 7 cells by microcellmediatedchromosome transfer (MMCT), and demonstrate that cells growtharrest before 10 to 12 population doublings. In contrast, theintroduction of a normal chromosome 13 had no effect upon growthof these cells either In vitro or In vivo. These data providedirect functional evidence for the presence of a growth suppressorgene(s) on chromosome 17, which is not p53, and which may representone of several gene(s) that play a critical role in the developmentof breast cancer.     

Designs for Instruction, Designs for Change: Distributing Knowledge of Evidence-Based Practice

We comment on the target article by Weingardt (this issue), which discusses recent advances in instructional design and technology (IDT) and their implications for dissemination of evidence-based practices. These arguments are extended to the topic of psychological intervention design, and possibilities for new intervention structures are briefly explored. Finally, comments are offered on maintaining a careful balance between technological and social processes in the effort to promote the dissemination of innovative and evidence-based psychological procedures.     

Porphyromonas gingivalis RgpA and Kgp proteinases and adhesins are C terminally processed by the carboxypeptidase CPG70

Porphyromonas gingivalis is a bacterial pathogen that produces the polyproteins RgpA and Kgp, which are proteolytically processed into proteinases and adhesins. We have demonstrated that the RgpA and Kgp proteinases and adhesins are C terminally processed by carboxypeptidase CPG70 by sequencing C-terminal peptides from both the wild type and an isogenic CPG70 mutant, using ion trap mass spectrometry.     

The FREP gene family in the snail Biomphalaria glabrata: additional members,and evidence consistent with alternative splicing and FREP retrosequences. Fibrinogen-related proteins

Fibrinogen-related proteins (FREPs) found in hemolymph of the snail Biomphalara glabrata are hypothesized to be involved in non-self recognition. Among 150 cloned FREP cDNAs examined, we have identified three additional FREP members, FREPs 3.3, 12.1 and 13.1, bringing the total of FREP subfamilies to 13. The new FREPs each encode two immunoglobulin superfamily domains and a fibrinogen domain. Additionally, five truncated cDNAs with >99% nucleotide identity in coding regions to FREPs 3.2, 12.1 or 13.1 were identified. The truncated forms, the first reported for FREPs, lack a partial exon, one complete exon, or two complete exons plus the 3'UTR. Our preferred hypothesis is that all five truncated cDNAs observed arise from alternative splicing of full-length FREP genes. Genomic sequences lacking at least two introns and corresponding to the 3' ends of the cDNAs of FREP12.1 and its two truncated forms were also recovered. Although these could be the source of the truncated cDNAs, they are believed to be retrosequences.     

Phosphoinositide deficiency due to inositol depletion is not a mechanism of lithium action in brain

The "inositol depletion hypothesis" has been widely held to be the explanation for both the effect of lithium on brain function, apropos of its use in mood disorders, and on the impairment of development and induction of embryonic malformations in diverse organisms. The essence of the hypothesis is that a deficiency in cellular myo-inositol (Ins), secondary to lithium inhibition of inositol monophosphatase and/or multiple inositol polyphosphate phosphatase activities with trapping of Ins as inositol phosphates, leads to a depression of phosphatidylinositol (PtdIns) and a secondary impairment in inositide signaling. However, the ability of relatively low micromolar levels of Ins to reduce mammalian PtdIns synthetase activity in vivo has never been adequately tested. We have generated a lethal murine brain Ins deficiency model and measured PtdIns content using a novel MALDI-TOF MS method. Our results show that in the most severe Ins deficiency ever recorded in a mammal, the brain PtdIns levels do not decrease. We conclude that PtdIns deficiency due to "inositol depletion" is not a mechanism of lithium action in brain, and that Ins plays another unidentified role in the mammalian brain.     

Immune response to lipopolysaccharide in primary biliary cirrhosis and autoimmune diseases

A bacteriological aetiology is suspected to be the triggering factor in primary biliary cirrhosis. We studied lipid A, the toxic and immunogenic moiety of gram-negative bacteria lipopolysaccharide, which accumulates abnormally in Kupffer cells, hepatocytes, and biliary epithelial cells in primary biliary cirrhosis patients. Anti-lipid A antibody levels from serum samples from 36 primary biliary cirrhosis patients, drawn before and after ursodeoxycholic acid treatment, were compared to those from patients with other liver diseases (n=236), non-hepatic diseases (n=249), and healthy subjects (n=75). In primary biliary cirrhosis patients, the prevalence of IgM anti-lipid A antibodies was higher before than after ursodeoxycholic acid therapy (64% vs 22%, respectively; P<0.001). Patients with anti-lipid A antibodies had significantly higher IgM levels than those without antibodies (8.7+/-1.1 g/l vs 4.4+/-0.8 g/l, P<0.02). Total IgM levels were correlated with anti-lipid A antibody levels (r=0.65, P<0.02). After therapy, the serum IgM levels decreased significantly (P<0.03). These results indicate that bacterial antigens may participate in the observed increase of serum IgM levels, and support an aetiological role of a gut-derived endotoxin antigen in the pathogenesis of primary biliary cirrhosis.     

Failure to produce conditioning with low-dose trimethylthiazoline or cat feces as unconditioned stimuli

Trimethylthiazoline (TMT), a derivative of fox feces, has been reported to fail to produce aversive conditioning as an unconditioned stimulus (UCS) when presented in large amounts (I. S. McGregor, L. Schrama, P. Ambermoon, & R. A. Dielenberg, 2002). Experiment I evaluated very low TMT levels that nonetheless produced defensive behaviors in rats during exposure. Although each level (0.01, 0.05, and 0.10 microl TMT) produced significant change in defensiveness, none resulted in significant changes the following day in the absence of TMT. Experiment 2 evaluated cat urine, cat feces, and cat fur/skin odor against a no-odor control. Urine produced no significant changes, but feces and fur/skin odors elicited virtually identical changes in defensive behaviors during exposure. When tested the next day in the absence of odor, the fur/skin odor-exposed group showed significant differences on the same behaviors as during exposure, but the feces-exposed group showed no differences on any measure. Results suggest that lack of conditioning to TMT may relate to the type of predator odor rather than the amount, predator species, or possible lack of odor components in TMT that are present in natural feces. Predator feces may also be less effective as a UCS because they are poorly predictive of the actual presence of the predator, suggesting the need for a reevaluation of UCS functions in aversive conditioning.