Dec 15, 2000. Cleavage at Asn66-Leu67 in the gelatinase A prodomain (29,30) by a free MT1-MMP molecule produces a 68-kDa activation intermediate (8, 12). Wells were then blocked with 2.5% (w/v) ovalbumin or 1% (w/v) BSA in PBS and serially diluted recombinant hemopexin C domain proteins then added.

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Original Article An Epidemic, Toxin Gene–Variant Strain of Clostridium difficile L. Clifford McDonald, M.D., George E. Killgore, Dr.P.H., Angela Thompson, M.M.Sc., Robert C. Owens, Jr., Pharm.D., Sophia V. Kazakova, M.D., M.P.H., Ph.D., Susan P. Sambol, M.T., Stuart Johnson, M.D., and Dale N.

Blast Effects On Buildings Pdf Free. One Up On Wall Street Free Ebook Pdf Reader. Gerding, M.D. N Engl J Med 2005; 353:2433-2441 DOI: 10.1056/NEJMoa051590.

Methods A total of 187 C. Difficile isolates were collected from eight health care facilities in six states (Georgia, Illinois, Maine, New Jersey, Oregon, and Pennsylvania) in which outbreaks of C. Difficile–associated disease had occurred between 2000 and 2003. The isolates were characterized by restriction-endonuclease analysis (REA), pulsed-field gel electrophoresis (PFGE), and toxinotyping, and the results were compared with those from a database of more than 6000 isolates obtained before 2001.

The polymerase chain reaction was used to detect the recently described binary toxin CDT and a deletion in the pathogenicity locus gene, tcdC, that might result in increased production of toxins A and B. Results Isolates that belonged to one REA group (BI) and had the same PFGE type (NAP1) were identified in specimens collected from patients at all eight facilities and accounted for at least half of the isolates from five facilities. REA group BI, which was first identified in 1984, was uncommon among isolates from the historic database (14 cases). Both historic and current (obtained since 2001) BI/NAP1 isolates were of toxinotype III, were positive for the binary toxin CDT, and contained an 18-bp tcdC deletion. Resistance to gatifloxacin and moxifloxacin was more common in current BI/NAP1 isolates than in non-BI/NAP1 isolates (100 percent vs.

42 percent, P. Figure 1 Major Genes in the Pathogenicity Locus (PaLoc) of Clostridium difficile and Relation to the Genes for Binary Toxin. Genes tcdA and tcdB encode toxins A and B, respectively, whereas tcdD encodes a positive regulator of the production of toxins A and B. Gene tcdE encodes a protein that may be important for the release of toxin from the cell. Gene tcdC is a putative negative regulator of the production of toxins A and B. Genes cdtA and cdtB are located at an unknown distance from the PaLoc and encode the enzymatic and binding components, respectively, of binary toxin.

B1 and A3 designate the location and relative size of the gene fragments that underwent polymerase-chain-reaction (PCR) amplification for toxinotyping. Clostridium difficile is a gram-positive, anaerobic, spore-forming bacillus that can cause pseudomembranous colitis and other C. Difficile–associated diseases. Studies during the 1970s showed that two toxins, A and B, were involved in the pathogenesis of C. Difficile–associated disease. Transmission occurs primarily in health care facilities, where exposure to antimicrobial drugs (the major risk factor for C. Difficile–associated disease) and environmental contamination by C.

Difficile spores are more common. Certain strains of C. Difficile have a propensity to cause outbreaks, including multistate outbreaks in health care facilities. Because these outbreak-associated strains are resistant to certain antimicrobial agents, such as clindamycin, the use of such antimicrobial agents provides these strains with a selective advantage over strains that are not associated with outbreaks. Historically low rates of severe disease and death (3 percent or less) may have led to an underestimation of the importance of C.

Difficile–associated disease as a health care–associated infection; however, each case of C. Difficile–associated disease has been estimated to result in more than $3,600 in excess health care costs, and these costs may exceed $1 billion annually in the United States. Both the rate and the severity of C.

Difficile–associated disease may be increasing in U.S. Health care facilities. An analysis of data from the National Nosocomial Infections Surveillance system identified an upward slope in C. Difficile–associated disease rates from the late 1980s through 2001. Of greater concern is a reported increase of 26 percentage points between 2000 and 2001 in the proportion of patients discharged from nonfederal U.S. Hospitals with C.

Difficile–associated disease listed as a diagnosis. Indications of the increased severity of C. Difficile–associated disease include reports from the University of Pittsburgh Medical Center, where the incidence of the disease in 2000 and 2001 was nearly twice as high as in 1990 through 1999. Twenty-six patients with severe disease required colectomy, and 18 patients died.

In addition, in the past two years, the Centers for Disease Control and Prevention (CDC) has received an increased number of reports from health care facilities of cases of severe C. Difficile–associated disease that have resulted in admissions to intensive care units, colectomies, and deaths. These reports have been confirmed by a nationwide survey of infectious-disease physicians in the Emerging Infections Network of the Infectious Diseases Society of America, which found that approximately 39 percent of respondents noted an increase in the severity of cases of C. Difficile–associated disease in their patient population. One explanation for an increase in both the rate and the severity of C.

Difficile–associated disease could be the emergence of an epidemic strain with increased virulence, antimicrobial resistance, or both. To examine this possibility, we characterized C. Difficile isolates obtained from health care facilities that reported outbreaks from 2001 through 2003 and compared these isolates with historic isolates (obtained before 2001) with the use of strain typing, identification of genetic determinants of newly described virulence factors, and testing for antimicrobial susceptibility. Health Care Facilities and Isolates from Patients Isolates were collected from patients in eight health care facilities that had reported an outbreak of C.

Difficile–associated disease since 2001 to investigators at either the CDC or the Hines Veterans Affairs (VA) Hospital. These facilities were located in six states (Georgia, Illinois, Maine, New Jersey, Oregon, and Pennsylvania); all were acute care hospitals, except for one long-term care facility in Georgia that was associated with a VA hospital. The isolates were obtained from patients who had received a diagnosis of C. Difficile–associated disease on the basis of clinical history (e.g., diarrhea with recent receipt of an antimicrobial drug) and a positive clinical laboratory test for C.

Difficile toxin (e.g., cytotoxin assay or enzyme immunoassay). Isolates from current (since 2001) outbreaks were compared with isolates from a historic (pre-2001) database of more than 6000 C. Difficile isolates maintained by Hines VA investigators. The isolates in the historic database were collected during the period from 1984 through 1990; all isolates were extensively characterized by HindIII restriction-endonuclease analysis (REA) and linked to clinical and epidemiologic data. Strain Typing The isolates underwent REA typing and pulsed-field gel electrophoresis (PFGE), as previously described; software from BioNumerics 3.5 (Applied Maths) was used to perform dendrographic analysis of the PFGE results. In addition, toxinotyping was performed according to the method of Rupnik et al., with modifications. Toxinotyping analyzes the restriction-fragment–length polymorphisms (RFLPs) of the genes encoding toxins A ( tcdA) and B ( tcdB), the surrounding regulatory genes ( tcdC and tcdD), and a porin gene ( tcdE) in a region of the C.

Difficile genome known as the pathogenicity locus (PaLoc) ( Figure 1 Major Genes in the Pathogenicity Locus (PaLoc) of Clostridium difficile and Relation to the Genes for Binary Toxin. Genes tcdA and tcdB encode toxins A and B, respectively, whereas tcdD encodes a positive regulator of the production of toxins A and B.

Gene tcdE encodes a protein that may be important for the release of toxin from the cell. Gene tcdC is a putative negative regulator of the production of toxins A and B. Genes cdtA and cdtB are located at an unknown distance from the PaLoc and encode the enzymatic and binding components, respectively, of binary toxin.

B1 and A3 designate the location and relative size of the gene fragments that underwent polymerase-chain-reaction (PCR) amplification for toxinotyping. Because RFLP analysis of polymerase-chain-reaction (PCR) fragments A3 and B1 results in a pattern sufficient to identify most toxinotypes, we limited our analysis to these two fragments. Molecular Markers of Potentially Increased Virulence In addition to the well-characterized A and B toxins, a binary toxin has been identified in about 6 percent of clinical C. Difficile isolates obtained in the United States and Europe. The structure and function of this toxin (referred to as binary toxin CDT) are similar to those of other binary toxins, such as the iota toxin found in C. Perfringens, and it is a suspected virulence factor in strains of C.

Difficile that carry the toxin. We detected the C. Difficile binary toxin gene by using PCR for cdtB, which is located outside the PaLoc and encodes the beta subunit of the binary toxin ( ). We also looked for deletions in tcdC by using PCR with the primers tcdc1 and tcdc2, which were synthesized at the CDC Core Facility on the basis of published sequences.

The gene tcdC is located within the PaLoc downstream from the genes encoding toxins A and B, and it is transcribed in the opposite direction from these genes ( ). The tcdC protein is thought to function as a negative regulator of the production of toxins A and B. Recently, multiple alleles of tcdC have been described that include different-sized deletions, point mutations, and in one case, a nonsense mutation, all of which would result in a truncated tcdC protein.

It has been hypothesized that mutations in tcdC may result in a loss of negative regulatory function, leading to increased toxin production and virulence. Testing for Antimicrobial Susceptibility Susceptibility to clindamycin and the fluoroquinolones (levofloxacin, gatifloxacin, and moxifloxacin) was determined with the use of E-test strips (AB Biodisk), and the results were interpreted according to standard criteria.

Specific breakpoints for the interpretation of clindamycin-susceptibility results were available from the Clinical and Laboratory Standards Institute (CLSI; formerly the National Committee for Clinical Laboratory Standards). However, because no breakpoints have been set by the CLSI for C. Difficile tested against these fluoroquinolones, the CLSI breakpoints for C. Difficile tested against trovafloxacin were used. The validity of the trovafloxacin breakpoints was confirmed by identification of two distinct subpopulations in the distribution of minimum inhibitory concentrations (MICs) for apparently susceptible isolates, as compared with resistant isolates, tested against these fluoroquinolones; these subpopulations were demarcated by the trovafloxacin breakpoints. Quality control of antimicrobial-susceptibility testing was performed during each test run with the standard strains Enterococcus faecalis American Type Culture Collection (ATCC) 29212, Pseudomonas aeruginosa ATCC 27583, Bacteroides fragilis ATCC 25285, and B.

Thetaiotaomicron ATCC 29741. Statistical Analysis To compare the overall resistance patterns of current epidemic and nonepidemic isolates, a total of three (determined according to the availability of isolates) epidemic-strain (case) and three nonepidemic-strain (control) isolates, as determined by REA and PFGE, were randomly selected from each health care facility. Resistance was then compared by matched case–control analysis with the use of Epi Info software (version 6.02). This method was chosen to take into account possible geographic variation in resistance and to avoid bias resulting from outbreaks with a larger number of isolates. In contrast, we used Fisher's exact test and the StatCalc function of Epi Info software (version 6.02) to make an unmatched comparison between current and historic epidemic isolates.

All P values are based on a two-tailed comparison. Results A total of 187 isolates were obtained from the eight health care facilities in which the outbreaks occurred. In each of the facilities, a strain composed of closely related isolates was identified by both PFGE and REA. This epidemic strain accounted for 50 percent or more of the isolates from five of the eight facilities ( Table 1 Isolates of Clostridium difficile According to Health Care Facility and the Proportion of Isolates Belonging to the BI/NAP1 Strain. The epidemic strain has been identified as belonging to REA group BI and North American PFGE type 1 (NAP1). Within this strain, characterized as BI/NAP1, the isolates have been further differentiated on the basis of minor differences in the band pattern into 14 REA subtypes, designated by numbers, in which at least 90 percent of the bands are identical.

Similarly, several PFGE subtypes are included in the NAP1 designation. Five REA BI types (BI1 through BI5), dating back to 1984, were identified in the historic database.

These represented 18 isolates obtained from 14 patients and consisted of 5 isolates of BI1 from 4 patients, 8 isolates of BI2 from 7 patients, 2 isolates of BI3 from 1 patient, 2 isolates of BI4 from 1 patient, and 1 isolate of BI5 from 1 patient. One isolate from each of the five REA BI types in the historic database was selected for further genetic testing, along with three BI/NAP1 and three non-BI/NAP1 current isolates from each health care facility.

The PFGE results and the dendrogram of these representative isolates are shown in Figure 2 Pulsed-Field Gel Electrophoresis Results and Dendrographic Analysis of a Sample of BI/NAP1 and Non-BI/NAP1 Isolates from Current Outbreaks of Clostridium difficile–Associated Disease and of Isolates from a Historic Database. The years listed for the historic isolates indicate years in which isolates of that type were recovered from patients, according to the database. The asterisk denotes the presence of a 39-bp deletion in tcdC., along with the toxinotype, the status of binary CDT, and the status of a deletion in the tcdC gene. According to dendrographic analysis, 25 of 29 of the combined current and historic BI/NAP1 isolates (86 percent) were 90 percent or more related, and all were more than 80 percent related. In contrast to this close relatedness among BI/NAP1 isolates across a wide geographic area, relatively few non-BI/NAP1 isolates were more than 80 percent related. All of the BI/NAP1 isolates were of toxinotype III, were positive for binary toxin CDT, and had an 18-bp deletion in tcdC; these features were largely absent among non-BI/NAP1 isolates ( ). Of the 24 non-BI/NAP1 isolates, 20 (83 percent) were toxinotype 0, none of which had binary toxin CDT or the tcdC deletion.

Susceptibility testing was performed on the 3 current BI/NAP1 and non-BI/NAP1 isolates from each health care facility, as well as on the 14 patient BI isolates available from the historic database. Among current isolates (obtained after 2000), all BI/NAP1 and only a fraction of the non-BI/NAP1 isolates were resistant to gatifloxacin and moxifloxacin ( Table 2 Resistance of Current BI/NAP1 Clostridium difficile Isolates, Current Non-BI/NAP1 Isolates, and Historic BI/NAP1 Isolates to Clindamycin and Fluoroquinolones. Although both BI/NAP1 and non-BI/NAP1 isolates were largely resistant to clindamycin and levofloxacin, the MICs of levofloxacin were higher for BI/NAP1 isolates as a group ( Figure 3 Distribution of Minimum Inhibitory Concentrations of Levofloxacin for Current (Obtained after 2000) BI/NAP1 and Non-BI/NAP1 Clostridium difficile Isolates. All current BI/NAP1 isolates and no historic isolates (obtained before 2001) were resistant to gatifloxacin and moxifloxacin ( ). Discussion An epidemic strain of C. Difficile has been associated with outbreaks of C.

Difficile–associated disease in eight health care facilities since 2001. This strain is the same as the strain responsible for recent outbreaks outside the United States. It is classified by REA typing as BI and by PFGE as NAP1, and is distinct from the J strain (REA type J7/9) that was responsible for outbreaks during the period from 1989 through 1992. Eighteen related isolates of the BI REA group, obtained from 14 known U.S. Difficile–associated disease that occurred between 1984 and 1993, were found in a database of more than 6000 isolates (representing more than 100 REA groups). According to PFGE dendrographic analysis, the majority of BI/NAP1 strain isolates (including historic BI isolates) were more than 90 percent related, and all were more than 80 percent related.

Although current BI/NAP1 isolates shared with historic BI isolates the putative virulence factors of binary toxin and an 18-bp deletion in tcdC, the current isolates were more likely to be resistant to fluoroquinolones. Therefore, the increasing use of fluoroquinolones in U.S.

Health care facilities may have provided a selective advantage for this epidemic strain and promoted its widespread emergence. The most compelling evidence of an increase in the severity of C. Difficile–associated disease in the United States is found in the reports from Pennsylvania Facility A, where an increase in both the number of cases and the severity of the disease was noted in 2000 and 2001.

In addition, there was evidence of higher white-cell counts and more severe disease in patients infected with BI/NAP1 strains than in those infected with non-BI/NAP1 strains at the Illinois facility in our study. Another report from a Connecticut hospital indicates an increase in the number of cases of severe disease necessitating colectomy during a recent outbreak associated with the BI/NAP1 strain. However, reports of other outbreaks, such as the outbreak in the Georgia long-term care facility included in our study, do not suggest increased disease severity. Even in the case of Pennsylvania Facility A, investigators were unable to find a significant association between the occurrence of severe C. Difficile–associated disease and infection with the outbreak strain (P=0.23). Therefore, other factors, such as underlying host susceptibility, prevailing practices of the use of antimicrobial agents or approaches to the treatment of C.

Difficile–associated disease, may have an important role in the causation of severe disease. The importance of binary toxin CDT as a virulence factor in C. Difficile has not been established; however, a similar toxin, iota toxin, is responsible for virulence in C. In previous reports, binary toxin CDT was found in only about 6 percent of C.

Difficile isolates; therefore, our finding that the prevalence of this toxin is much higher in isolates from outbreaks associated with increased morbidity suggests that it could, indeed, affect the severity of C. Difficile–associated disease. Previous studies have indicated that C. Difficile strains with binary toxin CDT nearly always have polymorphisms in the PaLoc.

Binary toxin CDT has been associated with several different toxinotype patterns; in our isolates, it was associated with toxinotype III, which was infrequently found in previous clinical surveys. Pseudomembranous colitis is more frequent among patients infected with C. Difficile of toxinotype III than among patients infected with C. Difficile of other toxinotypes, suggesting that this toxinotype is associated with increased severity of the disease. The importance of the 18-bp deletion in tcdC is currently unknown. Although tcdC is a proposed negative regulator of the production of toxins A and B, it is not known whether this 18-bp deletion would render a tcdC product nonfunctional and lead to increased production of toxins A and B.

A recent report, however, indicates that BI/NAP1 isolates in vitro do, indeed, produce toxins A and B in considerably greater quantities and at higher rates than non-BI/NAP1 isolates. Nonetheless, additional research on the effects of binary toxin CDT and of tcdC deletions on the severity of C.

Difficile–associated disease appears warranted. In addition to geographic variation in disease severity, there is variation in the role of particular fluoroquinolones as risk factors in these outbreaks. The outbreak in the Georgia long-term care facility occurred after a change in the formulary from levofloxacin to a C-8-methoxy fluoroquinolone, gatifloxacin. Gatifloxacin was an important risk factor for C. Difficile–associated disease among patients, and the outbreak resolved after a formulary switch back to levofloxacin. The authors hypothesized that the higher antianaerobic activity of gatifloxacin than of levofloxacin led to a greater alteration in bowel flora and that this, combined with resistance to fluoroquinolone in the prevailing C.

Difficile strain, contributed to the outbreak. Similarly, in Pennsylvania Facility B, the outbreak started within three months after a switch in the formulary from levofloxacin to a C-8-methoxy fluoroquinolone (moxifloxacin); the preliminary results of a case–control study identify moxifloxacin as a risk factor for C.

Difficile–associated disease during the outbreak. In Pennsylvania Facility A, C. Difficile–associated disease was associated with the use of levofloxacin, clindamycin, and ceftriaxone. However, a higher proportion of cases of C. Difficile–associated disease was associated with levofloxacin (31 percent) than with clindamycin (10 percent) or ceftriaxone (7 percent). The emergence of a previously uncommon strain of C. Difficile that is more resistant and potentially more virulent than other strains indicates a need for inpatient health care facilities in North America to track the incidence of C.

Difficile–associated disease. Clinical outcomes of patients with C. Difficile–associated disease should also be monitored, especially if an increase in rates is noted. If an increase in the proportion of severe cases is noted, special consideration should be given to the need for early diagnosis and treatment. Strict infection-control measures, including contact precautions, should be instituted for all patients with C. Difficile–associated disease. In contact precautions, the patient is placed in a room alone or with another patient with C.

Difficile–associated disease, health care workers wear gloves and gowns when entering the room, and patient-care equipment (such as blood-pressure cuffs and stethoscopes) either is used only for the patient or is cleaned before it is used for another patient. Enhanced environmental cleaning with dilute bleach should be used to eliminate C. Difficile spores. Because alcohol is ineffective in killing C.

Difficile spores, it is prudent for health care workers to wash their hands with soap and water, rather than with alcohol-based waterless hand sanitizers, when caring for patients with C. Difficile–associated disease during an outbreak. Finally, an important method of controlling past outbreaks of C.

Difficile–associated disease has been restriction of the use of antimicrobial agents implicated as risk factors for the disease. Whether a large-scale restriction of the use of these antimicrobial agents could slow the geographic spread of the BI/NAP1 strain is not known.

Because fluoroquinolones have become a mainstay in the treatment of several common infections, a large-scale restriction of the use of these drugs would be quite difficult. However, if this epidemic strain continues to spread and to contribute to increased morbidity and mortality, it will be important either to reconsider the use of fluoroquinolones or to develop other innovative measures for controlling C. Difficile–associated disease. Presented in part at the 42nd Annual Meeting of the Infectious Diseases Society of America, Boston, October 1–3, 2004. Supported in part by grants from the Department of Veterans Affairs Research Service (to Drs.

Johnson and Gerding). Owens reports having received research funding from Elan, Bayer, Ortho-McNeil, and Pfizer; Dr. Johnson, research funding from Salix Pharmaceuticals and consulting fees or fees for service on an advisory board from Genzyme, Acambis, ViroPharma, and Salix Pharmaceuticals; and Dr. Gerding, research funding from Presutti Laboratories, ActivBiotics, Oscient Pharmaceuticals, and Optimer Pharmaceuticals and consulting fees or fees for serving on an advisory board from Acambis, Oscient Pharmaceuticals, ViroPharma, Genzyme, Optimer Pharmaceuticals, and Salix Pharmaceuticals. Gerding holds U.S., Canadian, and European Union patents for the use of nontoxigenic C. Difficile to treat and prevent C.

Difficile infection. We are indebted to the members of the Clostridium difficile Investigation Team for making isolates available for study or otherwise assisting in the characterization and analysis of isolates: Jemelae Bessette, Priscilla Biller, Adam Cheknis, Robert Gaynes, Carol Genese, Kathleen Gensheimer, David Gilbert, Lee Harrison, Bette Jensen, Susan Kohlhepp, James Martin, Linda McDougal, Michelle Merrigan, Carlene Muto, Gary Noskin, Sandra Reiner, Corey Robertson, Kathleen Roye-Horn, Steve Sears, Farida Siddiqui, Sarah Slaughter, Lisa Tkatch, Marty Topiel, August J. Valenti, Carol Ward, Kim Ware, John Warren, Lois Wiggs, Teresa Zembower, and Walter Zukowski. Source Information From the Epidemiology and Laboratory Branch, Division of Healthcare Quality Promotion, Centers for Disease Control and Prevention, Atlanta (L.C.M., G.E.K., A.T., S.V.K.); the Departments of Pharmacy and Infectious Diseases, Maine Medical Center, Portland (R.C.O.); the College of Medicine, University of Vermont, Burlington (R.C.O.); and the Infectious Disease Section and Research Service, Department of Medicine, Hines Veterans Affairs Hospital and Loyola University Stritch School of Medicine, Hines, Ill.

(S.P.S., S.J., D.N.G.). Address reprint requests to Dr. McDonald at 1600 Clifton Rd., MS A35, Atlanta, GA 30333,.

References • 1 Bartlett JG. Antibiotic-associated diarrhea. N Engl J Med 2002;346:334-339 • 2 Bartlett JG, Onderdonk AB, Cisneros RL, Kasper DL.

Clindamycin-associated colitis due to a toxin-producing species of Clostridium in hamsters. J Infect Dis 1977;136:701-705 • 3 Bartlett JG, Chang T, Taylor NS, Onderdonk AB. Colitis induced by Clostridium difficile.

Rev Infect Dis 1979;1:370-378 • 4 Taylor NS, Bartlett JG. Partial purification and characterization of a cytotoxin from Clostridium difficile. Rev Infect Dis 1979;1:379-385 • 5 Taylor NS, Thorne GM, Bartlett JG.

Comparison of two toxins produced by Clostridium difficile. Infect Immun 19-1043 • 6 Johal SS, Hammond J, Solomon K, James PD, Mahida YR. Clostridium difficile associated diarrhoea in hospitalised patients: onset in the community and hospital and role of flexible sigmoidoscopy.

Gut 2004;53:673-677 • 7 Johnson S, Samore MH, Farrow KA, et al. Epidemics of diarrhea caused by a clindamycin-resistant strain of Clostridium difficile in four hospitals. N Engl J Med 1999;341:1645-1651 • 8 Rubin MS, Bodenstein LE, Kent KC. Severe Clostridium difficile colitis.

Dis Colon Rectum 1995;38:350-354 • 9 Kyne L, Hamel MB, Polavaram R, Kelly CP. Health care costs and mortality associated with nosocomial diarrhea due to Clostridium difficile. Clin Infect Dis 2002;34:346-353 • 10 Archibald LK, Banerjee SN, Jarvis WR.

Secular trends in hospital-acquired Clostridium difficile disease in the United States, 1987-2001. J Infect Dis 2004;189:1585-1589 • 11 McDonald LC, Banerjee S, Jernigan DB. Increasing incidence of Clostridium difficile-associated disease in U.S. Acute care hospitals, 1993-2001. In: Proceedings of 14th Annual Scientific Meeting of the Society for Healthcare Epidemiology of America, Philadelphia, April 17-20, 2004. • 12 Dallal RM, Harbrecht BG, Boujoukas AJ, et al.

Fulminant Clostridium difficile: an underappreciated and increasing cause of death and complications. Ann Surg 2002;235:363-372 • 13 Muto CA, Pokrywka M, Shutt K, et al. A large outbreak of Clostridium difficile-associated disease with an unexpected proportion of deaths and colectomies at a teaching hospital following increased fluoroquinolone use.

Infect Control Hosp Epidemiol 2005;26:273-280 • 14 McEllistrem MC, Carman RJ, Gerding DN, Genheimer CW, Zheng L. A hospital outbreak of Clostridium difficile disease associated with isolates carrying binary toxin genes. Clin Infect Dis 2005;40:265-272 .

Abstract The mitogen-activated kinase activating death domain protein (MADD) that is differentially expressed in neoplastic vs. Normal cells (DENN) was identified as a substrate for c-Jun N-terminal kinase 3, the first demonstration of such an activity for this stress-activated kinase that is predominantly expressed in the brain. A splice isoform was identified that is a variant of MADD. A protein identical to MADD has been reported to be expressed differentially in neoplastic vs.

Normal cells and is termed “DENN.” We demonstrated differential effects on DENN/MADD in a stressed vs. Basal environment. Using in situ hybridization, we localized both the substrate and the kinase to large pyramidal neurons in the human hippocampus. It was interesting that, in four of four patients with neuropathologically confirmed acute hypoxic changes, we detected a unique translocation of DENN/MADD to the nucleolus.

These changes were apparent only in neurons sensitive to hypoxia. Moreover, in those cells, translocation of the substrate was accompanied by nuclear translocation of JNK3. These findings place DENN/MADD and JNK in important hypoxia insult-induced intracellular signaling pathways. Our conclusions are important for future studies for understanding these stress-activated mechanisms.

The c-Jun N-terminal kinases (JNKs), or stress-activated kinases, belong to the mitogen-activated kinase family sharing sequence homology with other members, including the extracellular signal-regulated kinases (ERKs). Three JNKs have been identified: JNK1, 2, and 3 (for reviews, see refs. Each JNK can be spliced differentially, yielding two to four isoforms depending on the tissues and species, and with varying C termini and internal substitutions of the putative c-Jun recognition and binding region (, ). Unlike JNK1 and 2, which are expressed ubiquitously in a variety of human tissues, JNK3 is found predominantly in the brain within neurons (). Developmentally, it is expressed in postmitotic neurons undergoing differentiation (). As with the ERKs, activation of JNK requires dual phosphorylation of both a threonine and a tyrosine residue located in a consensus tripeptide sequence.

Several substrates for JNK have been identified, such as c-Jun, ATF-2, Elk1, and p53 (–). Phosphorylation of these substrates results in elevated transcriptional activity (,,, ). Each JNK isoform binds the various substrates with different affinities. It is not known whether individual JNKs respond to distinct extracellular signals or lead to different downstream effects by preferentially phosphorylating specific substrates. JNK activity can be elevated by a variety of stimuli, including environmental stress (–), apoptotic agents (), or neurotoxic insults (). A kinase cascade immediately upstream of JNK leads to its induction (–), reminiscent of ERK activation, which involves a parallel mechanism. It is possible that mitogenic and stress signals are transduced by ERK and JNK, respectively, through their selective phosphorylation of different transcription factors.

Evidence for the direct involvement of JNK in apoptosis comes from several studies in PC12 cells; overexpression of a constitutively activated JNK kinase potentiates apoptosis induced by nerve growth factor (NGF) deprivation (). Conversely, microinjection of a c-Jun-dominant negative mutant into rat sympathetic neurons protects the cells from apoptosis (). In addition, increased c-Jun activity alone in NIH 3T3 fibroblasts is sufficient to trigger cell death (). Based on the expression pattern of JNK3 restricted to neurons and the established role for JNK in cell death pathways under stress, we explored the possibilities of neuron-specific functions of JNK3 beyond the general effects of c-Jun phosphorylation. We used the yeast two-hybrid system to search for novel proteins that interact with JNK3.

One such protein that proved to be a splice variant of the mitogen-activated kinase activating death domain protein (MADD) that is differentially expressed in neoplastic vs. Normal cells (DENN) revealed a relationship between JNK3 activation and the neuronal stresses of hypoxia/ischemia and the inflammatory response in the human central nervous system (CNS). Yeast Two-Hybrid Library Screening. The matchmaker two-hybrid system 2 from CLONTECH was used. The bait construct was generated as follows: two PCR primers were designed (primers were made by the Molecular Core Facility at the University of Southern California), the 5′ primer GTT ACC CGG GG A TGA GCC TCC ATT TCT TAT AC and the 3′ primer TAT GGT CGA CC A CTC TCA CTG CTG CTG TTC ACT G. The underlined sequences from the two primers were complementary to the nucleotides 231–251 and nucleotides 1494–1513 of JNK3 cDNA, respectively. The amplified JNK3 cDNA was inserted in-frame with the Gal4 DNA binding domain.

Successful construction of the fusion protein was verified on Western blots by using both anti-JNK antibody (Santa Cruz Biotechnology) and anti-Gal4-binding domain antibody (CLONTECH). The construct was then cotransformed into yeast cells with a human brain cDNA library fused to the Gal4 activation domain (Gal4-AD), and the cells were screened for colonies that activated the reporter genes LacZ and His3.

Plasmids were purified from yeast as described by Ward et al. () and transformed into Max efficiency-competent DH5α (GIBCO/BRL).

Automated plasmid sequencing was performed by the Molecular Core Facility at the University of Southern California School of Medicine. Construction of Glutathione S-Transferase (GST) 1.1 and Hemagglutinin (HA)–DENN/MADD. Clone 1.1 was subcloned into the XhoI and EcoRI sites of pGEX-4T-2 (Pharmacia). Four reverse transcription-PCR fragments were obtained, representing fragments of the full length DENN (using the numbering system by Chow and Lee, GenBank accession no. ): from nucleotide 165 to 1543, from nucleotide 1149 to the alternative splice junction in clone 1.1, from the splice junction to nucleotide 4054, and nucleotides 3920 to 4947. The most 5′ fragment was PCR-amplified again, adding an HA epitope (YPYDVPDYA) following the start codon.

These fragments and clone 1.1 were ligated and subcloned into pcDNA3 (Invitrogen, Carlsbad, CA). Cell Cultures. Mouse neuroblastoma Neuro-2A cells were obtained from American Type Culture Collection, routinely cultured in MEM (Sigma) and transfected by using lipofectAmine (GIBCO/BRL). Cells were harvested 2 days posttransfection, and the postnuclear supernatant was analyzed by Western blotting or immunoprecipitation by using antibodies including anti-HA (Boehringer Mannheim) and anti-DENN/MADD peptide antibodies and detected with horseradish peroxidase-conjugated secondary antibody (Transduction Laboratories, Lexington, KY) and the enhanced chemiluminescence method (Amersham). Human CNS tissues were obtained postmortem from five neurologically and histologically normal control patients with no infarction or agonal hypoxia and from four patients with known premortem hypoxia and with histological confirmation of hypoxic nerve cell changes. Based on the hematoxylin and eosin stain, these include a spectrum of changes starting with condensed, eosinophilic cytoplasm followed by nuclear pyknosis and neutrophil and macrophage infiltration. Tissues (1 cm 3) were snap-frozen in liquid nitrogen-chilled isopentane and stored at −90°C.

The postmortem interval of the tissues from both sets of patients ranged from 2 to 8 h. Cryostat sections (10 μm) of hippocampus or cerebellum, the most hypoxia-sensitive regions, or Neuro2A cells cultured on Biocoat chamber slides (Becton Dickinson) were used. Slides were air dried and fixed in ice-cold acetone. After blocking with 5% goat serum, primary antibodies including rabbit anti-DENN/MADD C-terminal antibody or rabbit anti-JNK3 antibody (Upstate Biotechnology, Lake Placid, NY) were each diluted 1:100 and allowed to incubate at room temperature for 1 h.

After washing with PBS, a biotinylated goat anti-rabbit IgG secondary antibody (Vector Laboratories) was added, and the avidin–biotin complex (Vector Laboratories) and AEC (Zymed) methods were used for visualization. Sections were counterstained with Mayer’s hematoxylin solution (Sigma). Yeast Two-Hybrid Screening and Expression of DENN/MADD in the CNS. Using the yeast two-hybrid system (CLONTECH), ≈1.5 × 10 6 colonies were screened in a human brain cDNA library, and 41 positive clones were obtained that activated transcription of the reporter genes. These clones did not interact with the Gal4-AD itself nor with Gal4-AD fused to either p53 or lamin.

Sequence analysis revealed that one clone, clone 1.1, represented a partial human cDNA of DENN (submitted to GenBank by Chow and Lee, accession no. As illustrated in Fig.

A, the 370 amino acids encoded by clone 1.1 were identical to those of DENN, with the important exception that amino acids 762–804 were absent from the yeast clone, suggesting the possibility of a splice variant. Figure 1 Cloning of DENN/MADD and its splice variant. ( A) Graphic representation of clone 1.1 and human DENN/MADD. Sequence comparison with C. Elegans homolog AEX-3 also is illustrated.

Shaded boxes are regions conserved between the two proteins. Three motifs in human are shown, one of which also occurs in AEX-3. ( B) Northern analysis. A human tissue mRNA blot (CLONTECH) was used with clone 1.1 as the probe. Hybridization of the same blot with an actin cDNA probe indicated that similar amounts of RNA were present in each lane (data not shown).

( C) Reverse transcription-PCR of DENN/MADD. MRNA (1 μg) from human brain was reverse-transcribed and amplified by using the primers illustrated in A: PCRI, nucleotides 2334–2689; PCRII, nucleotides 2334–3024. In both PCR reactions, multiple bands are seen.

Clone 1.1 corresponds to the lower band in PCRII. To determine whether clone 1.1 was contiguous with the remainder of the DENN sequence, primers were designed to encompass the junction of the putative splice site and were hybridized only with the spliced form consistent with clone 1.1. Reverse transcription followed by PCR amplification of human brain mRNA was carried out (Fig. A, reactions 1 and 2). Sequencing of both PCR products confirmed that clone 1.1 was a spliced form of DENN in the human brain. In addition, our data revealed an additional G at nucleotide 4022, upstream from the original stop codon assigned by Chow and Lee at nucleotide 4041. This frameshift results in utilization of the stop codon at nucleotide 4940 and translation of a polypeptide 300 residues longer.

Recently, cloning of the mitogen-activated kinase-activating death domain protein (MADD) that interacts with the tumor necrosis factor receptor (TNFR) was reported (31, GenBank accession no. The MADD sequence is virtually identical to that of DENN. Consistent with our results, MADD includes the C-terminal 300-amino acid extension. We will therefore refer to the clone used as DENN/MADD.

Examination of the primary sequence of human DENN/MADD reveals two leucine zippers, a putative nuclear localization signal (PRGKRRAK) and a potential proline-rich SH3 binding domain (KPLPSVPP) () (Fig. The SH3 binding domain is also present in clone 1.1, adjacent to the variably spliced site. Homologs of human DENN/MADD have been isolated from rat and Caenorhabditis elegans as important mediators of regulated neurotransmitter release. The rat DENN/MADD shares >90% identity with its human counterpart, whereas the C. Elegans homolog (AEX-3) exhibits various degrees of similarity in three domains, with extensive variability in the rest of the sequence (Fig.

In addition, the putative nuclear localization signal and the SH3 binding domain, as well as one of the leucine zippers, are missing from AEX-3. To determine the expression pattern of DENN/MADD in human tissues, Northern analysis was performed. A transcript of ≈7.2 kb was evident in heart, brain, placenta, muscle, and pancreas but was absent in lung, liver, and kidney (Fig. The mRNA species detected in the brain appeared to be homogeneous. To determine whether one or both of the two splice variants were represented, primers flanking the putative splice site were used in reverse transcription-PCR reactions. As shown in Fig. C, PCRII, two bands of 355 and 226 bp were amplified, consistent with the predicted sizes of the fragments derived from DENN/MADD and clone 1.1, respectively.

Sequencing of the two bands confirmed that the 226-bp band corresponded to clone 1.1, whereas the broader, 355-bp band was a mixture of the original DENN/MADD and another sequence (not shown). Of interest, a similar reaction, using primers outside of those used in PCRII (Fig. C, PCRI), yielded multiple bands, indicating the existence of additional splice isoforms. Thus, at least three splice variants are present in the adult human brain, although their functional differences remain to be elucidated. It is likely that the 129-bp difference in size between DENN/MADD and clone 1.1 was too small to be distinguishable on the Northern blot. Figure 2 Phosphorylation of GST-1.1. ( A) GST-1.1 expressed in Escherichia coli was purified and incubated with 1, 2, or 3 μl of JNK3 in the presence of γ- 33P-ATP at 30°C for 20 min.

Reactions were stopped by the addition of SDS sample buffer and resolved on the gel (lanes 2, 3, and 4). The asterisk (∗) indicates the phosphorylated GST-1.1. GST–c-Jun (1 μg) is shown in lane 1 as a positive control. ( B) Comparison of phosphorylation of GST-1.1, myelin basic protein, and GST–c-Jun by three different kinases: C (p34 cdc2/cyclinB), E (ERK2), or J (JNK3). Note that GST-1.1 is phosphorylated preferentially by JNK3 (lane 3).

Expression of Full Length DENN/MADD in Neuro2A Cells. HA-tagged, full length DENN/MADD was constructed with the HA epitope added at the N terminus of the fusion protein. Expression of the HA–DENN/MADD in Neuro2A cells was confirmed by Western blot by using a rabbit polyclonal antibody generated against a C-terminal peptide of DENN/MADD.

As shown in Fig. A, a band of ≈200 kDa corresponding to the protein expressed by the full length cDNA was recognized by both anti-HA and anti-peptide antibody only in the transfected samples (Fig. A, lanes 1 and 3). To demonstrate the specificity of the anti-DENN/MADD antibody for immunostaining, mock- (no DENN/MADD cDNA transcript) or human DENN/MADD cDNA-transfected Neuro2A cells grown on chamber slides were examined with the antibody. Brightly stained cells were only apparent in the cDNA-transfected cells (Fig. B) but not in the negative control (not shown).

Figure 3 Expression of full length DENN/MADD in Neuro2A cells. ( A) Transfected with either pcDNA3 (lanes 2 and 4) or HA-tagged DENN/MADD cDNA in pcDNA3.

Western blots are shown that included either anti-HA antibody (lanes 1 and 2) or anti-DENN/MADD peptide antibody (lanes 3 and 4). DENN/MADD appears as a band of ≈200 kDa in lanes 1 and 3.

( B) Cells transfected with DENN/MADD cDNA were analyzed for DENN/MADD expression by immunostaining by using the anti-peptide antibody. Arrowhead points to a nontransfected cell; arrow indicates a transfected cell that shows diffuse cytoplasmic staining. (Bar = 10 μm.). Co-Localization of JNK3 and DENN/MADD by in Situ Hybridization. One prerequisite for JNK3 to phosphorylate DENN/MADD in vivo is that they be expressed in the same cell type. In situ hybridization was used to detect the mRNA distribution of the kinase and its substrate in the human hippocampus.

Shows abundant silver grains with both antisense probes compared with the sense probes, indicating the presence of both JNK3 and DENN/MADD mRNA (Fig. D) in large neurons in the hippocampal CA4 region. Similar results also were observed in pyramidal neurons in the CA1 region and subiculum (data not shown). Localization of DENN/MADD by Immunohistochemistry in the Normal and Hypoxic Nervous System. Immunohistochemical studies were performed on cryostat sections obtained from brain tissues obtained postmortem from four normal patients. As shown in Fig. A, JNK3 immunostaining was sparse and diffusely distributed in the cytoplasm of pyramidal neurons in the hippocampus.

These neurons also expressed scant to moderate amounts of DENN/MADD in their somas (Fig. Preimmune sera gave none of the signals (data not shown). In the cerebellum, DENN/MADD was found in the Purkinje cell cytoplasm (Fig. D) and in the strongly stained processes and terminals extending transversely across the molecular layer nearly to the pial surface (Fig. In contrast, in the hippocampi of four patients with typical histological evidence of acute hypoxia including condensed, eosinophilic cytoplasm and with neuronophagia (macrophage invasion), (Fig.

G), JNK3 was shown to be more strongly expressed, especially in the nucleus (Fig. Concomitantly, anti-DENN/MADD staining also was modified by striking labeling of the nucleolus (Fig. This nuclear and nucleolar staining of DENN/MADD only was observed in the CA1 region and subiculum, regions known to be selectively sensitive to hypoxia (Fig. In two of two other patients with cerebellar hypoxic changes, some Purkinje cells also showed nucleolar staining of DENN/MADD (Fig. This was not observed in any of the five normal control patients whose brain lacked signs of hypoxia/ischemia.

Figure 5 Immunohistochemical localization of JNK3 and DENN/MADD. Cryostat sections of normal ( A– D) or hypoxic/ischemic brain ( E– H). ( A) In normal hippocampus (CA-1) stained with anti-JNK3, there is minimal, diffuse cytoplasmic staining of pyramidal neurons. ( E) In contrast, hippocampus with hypoxic change shows strong nuclear immunoreactivity. Anti-DENN/MADD antibody shows weak to moderate cytoplasmic staining of normal hippocampal pyramidal neurons in B. ( G) The hypoxic/ischemic hippocampus shows typical eosinophilic pyramidal neurons. Note the perineuronal macrophages (hematoxylin and eosin).

( F) Anti-DENN/MADD immunostain of a comparably affected hippocampus shows intense nucleolar stain. In the normal cerebellum in D, Purkinje cells show diffuse cytoplasmic staining. ( C) There is also intense staining of neurites in the molecular layer of the normal cerebellum. In the hypoxic/ischemic cerebellum, there is strong nucleolar staining of the Purkinje cells in H.

(Bar = 25 μm in A, E, and G; bar = 10 μm in B and F; and bar = 2.5 μm in C, D, and H. DISCUSSION We report the identification and sequencing of a new splice variant, GST-1.1, of human DENN/MADD.

Recombinant GST-1.1 was phosphorylated preferentially by JNK in vitro but not by p34 cdc2/cyclinB or ERK, suggesting that DENN/MADD is a favored substrate for JNK. DENN/MADD is heavily phosphorylated, and JNK is the first kinase accounting for this function ().

There are five Ser-Pro and one Thr-Pro sequences in clone 1.1 that potentially serve as acceptor sites for JNK. The actual residues used have not been identified yet. Although c-Jun and ATF-2 both bind JNK with considerable affinity in vitro (, ), DENN/MADD failed to do so with JNK, either in the coimmunoprecipitation or the in vitro pull-down assay (data not shown), despite the fact that the DENN/MADD variant was discovered in the yeast screen. () hypothesized that binding and phosphorylation are separate attributes of JNK.

At least two other established substrates, Elk-1 and JunD, do not exhibit detectable in vitro binding to JNK. We generated two truncated JNK3 cDNA constructs that encompassed either the N terminus (residues 1–245) or the C terminus (residues 147–381).

Both fragments contained the corresponding region in JNK2 previously reported to be essential for efficient c-Jun binding (). However, neither of these truncated JNK3 forms interacted with clone 1.1 in the yeast system (data not shown), suggesting that the entire JNK sequence may be required for measurable binding to occur, consistent with results obtained by Gupta et al. () for other substrates. Future mutagenesis studies may determine precisely which residues in clone 1.1, including the SH3 binding domain, are important for JNK recognition. Our data indicated that JNK phosphorylated DENN/MADD with significantly higher activity than did ERK. In addition to JNK, the C terminus of DENN/MADD (not overlapping with clone 1.1) also interacted with TNFR1 as part of the TNFR signaling complex (). Schievella et al.

() demonstrated that, in COS cells, overexpression of a partial cDNA starting at F1269, but not the full length DENN/MADD, strongly induces JNK activity independently of TNF. In contrast, ERK activity is augmented by full length DENN/MADD and TNF has an additive effect. Concomitant activation of ERK and JNK has been documented to occur with TNFα acting through either TNFR1 or TNFR2 (, ), although ERK and JNK appear to have opposing effects on cellular growth and death. For example, ERK activation counteracts Fas- or nerve growth factor deprivation-induced/JNK-mediated apoptosis (, ). In this scenario, ERK is the principal effector of the TNF–TNFR1–DENN/MADD pathway.

Subsequently, TNF stimulates JNK, probably by other pathways (), which, in turn, phosphorylates DENN/MADD. How, then, does phosphorylation of DENN/MADD impact on its activation of the mitogen-activated kinases? We speculate that phosphorylated DENN/MADD may suppress the ERK pathway when the JNK pathway is active. TNFα also induces a mitogen-activated protein kinase kinase kinase, ASK1, with apoptosis as the consequence (). It remains to be examined whether DENN/MADD is involved in the action of ASK1 as well as the physiological consequences of TNFα induction of JNK.

DENN/MADD homologs have been cloned from rat and C. Elegans as GDP/GTP exchange proteins specific for the Rab3 subfamily members to regulate exocytosis of neurotransmitters (, ). Consistent with such a function in vesicle release, we observed expression of DENN/MADD in the neuronal processes in the cerebellum and hippocampus (Fig. In the latter site, Rab3 is essential for long term potentiation ().

It is yet unknown whether phosphorylation alters the GTP/GDP exchange activity of DENN/MADD. Immunohistochemical localization of DENN/MADD in normal human CNS tissues revealed focal cytoplasmic immunoreactivity of neuronal subpopulations, including their proximal and distal neurites.

In all four patients with either acute hypoxic cell change, including with neuronophagia, the cytoplasmic DENN/MADD immunostaining showed both enhanced expression and an altered subcellular distribution. Striking nucleolar staining was apparent in neurons of the hypoxia-sensitive regions of the hippocampus, including CA1 and the subiculum, as well as in Purkinje cells in the cerebellum.

These changes reflect the nonuniform susceptibility of neurons to hypoxia/ischemia in the CNS. There is a well established hierarchy of selective sensitivity among neurons throughout the nervous system. Most susceptible are pyramidal neurons of the subiculum, CA1 regions of the hippocampus, and Purkinje cells of the cerebellum.

Microscopically, a fairly constant time course evolves after the hypoxic/ischemic insult. The earliest and characteristic hallmark, visible after 4 h, is the eosinophilic or “red neurons” followed by nuclear pyknosis and disappearance of the nucleoli.

By 15–24 h, neutrophils infiltrate the tissues, and by 2–3 days, there is an influx of macrophages. Thus, the association of DENN/MADD with the nucleolus is a change that happens very early, perhaps when the damage is still reversible. As a stress-activated kinase, JNK3 presumably would be activated under the hypoxic conditions in these neurons.

Of interest, expression of JNK3 in the hippocampus also was found to be increased, in particular in the nucleus (Fig. F), placing the kinase and substrate in the same intracellular compartment, consistent with our hypothesis for their interaction.

These observations support the results of Dragunow et al. (), who report that hypoxia/ischemia increased c-Jun expression in CA1 of the rat hippocampus mediated by N-methyl- d-aspartate. In our experiments, when Neuro2A cells were transfected with DENN/MADD cDNA, the expressed protein was concentrated in the cytoplasm, suggesting that exogenous signals may be necessary for translocation to the nucleolus to occur. Because our antibody recognizes the C terminus of DENN/MADD that is shared among various splice isoforms, it is unclear presently whether one or all isoforms are translocated into the nucleus. A growing list of nuclear proteins have been found to be recruited into the nucleolus. This diverse array includes the tumor suppressor Rb (), transcription factor YY1 (), heat-shock protein 70 (), histone deacylase (), TATA-binding protein (), some viral proteins such as HIV Rev (), and herpesvirus MEQ (). Although a consensus sequence has yet to be defined, the nucleolar localization signal generally involves a stretch of positively charged residues.

Functionally, these proteins may influence DNA replication or transcriptional activity. The role of DENN/MADD in nucleolar function remains unclear, but the restricted pattern concentrated in the hypoxia-sensitive regions suggests that it may be part of a neuronal injury response. As a stress-activated kinase, JNK3 potentially is induced under these conditions, and activated JNK has been shown to be targeted to the nucleus (refs. It is tempting to speculate that, after translocation of both the substrate and kinase transducing the stress signal in the hypoxia-sensitive neurons, JNK3 then phosphorylates DENN/MADD. We propose that DENN/MADD has dual activities: Normally, it modulates neurotransmitter release of a subset of neurons functioning as a “housekeeping” gene evolutionarily conserved from C. Elegans to human.

This function is likely to be independent of the phosphorylation state because the domain important for JNK recognition (clone 1.1) is localized in the divergent region between C. Elegans and human (Fig.

In addition, JNK may be bound tightly to a cytoplasmic anchoring inhibitor () and therefore be unavailable for DENN/MADD phosphorylation. Under stress conditions, such as hypoxia or macrophage invasion, DENN/MADD may translocate into the nucleolus. Elucidation of the complex pathway of DENN/MADD and JNK could help to understand how neurons respond to stress and could provide insight into nucleolar regulatory mechanisms. Acknowledgments We greatly appreciate the allocation of autopsied brain tissues by the Alzheimer’s Disease Research Center at University of Southern California. We also thank Celia Williams, Ning Sun, Zhiqun Tan, and Steven Schreiber for technical advice on in situ hybridization and immunohistochemistry and Bernard Freidin for his computer expertise. This work was supported in part by the National Institute of Mental Health (5R37MH39145), the National Institute on Aging (5P50AG05142), the Sankyo Corporation (Sankyo) to C.A.M., and National Institutes of Health training grant (5K12AG00521) to Y.Z.