FRA-1 as a driver of tumour heterogeneity: a nexus between oncogenes and embryonic signalling pathways in cancer.

Tumour heterogeneity is a major factor undermining the success of therapies targeting metastatic cancer. Two major theories are thought to explain the phenomenon of heterogeneity in cancer--clonal evolution and cell plasticity. In this review, we examine a growing body of work implicating the transcription factor FOS-related antigen 1 (FRA-1) as a central node in tumour cell plasticity networks, and discuss mechanisms regulating its activity in cancer cells. We also discuss evidence from the FRA-1 perspective supporting the notion that clonal selection and cell plasticity represent two sides of the same coin. We propose that FRA-1-overexpressing clones featuring high plasticity undergo positive selection during consecutive stages of multistep tumour progression. This model underscores a potential mechanism through which tumour cells retaining elevated levels of plasticity acquire a selective advantage over other clonal populations within a tumour.

PR55α-containing protein phosphatase 2A complexes promote cancer cell migration and invasion through regulation of AP-1 transcriptional activity.

The proto-oncogene c-Jun is a component of activator protein-1 (AP-1) transcription factor complexes that regulates processes essential for embryonic development, tissue homeostasis and malignant transformation. Induction of gene expression by c-Jun involves stimulation of its transactivation ability and upregulation of DNA binding capacity. While it is well established that the former requires JNK-mediated phosphorylation of S63/S73, the mechanism(s) through which binding of c-Jun to its endogenous target genes is regulated remains poorly characterized. Here we show that interaction of c-Jun with chromatin is positively regulated by protein phosphatase 2A (PP2A) complexes targeted to c-Jun by the PR55α regulatory subunit. PR55α-PP2A specifically dephosphorylates T239 of c-Jun, promoting its binding to genes regulating tumour cell migration and invasion. PR55α-PP2A also enhanced transcription of these genes, without affecting phosphorylation of c-Jun on S63. These findings suggest a critical role for interplay between JNK and PP2A pathways determining the functional activity of c-Jun/AP-1 in tumour cells.

Avian mycobacteriosis in psittacines: a retrospective study of 123 cases.

One hundred and twenty-three cases of mycobacterioses were diagnosed in psittacine birds from a total of 9,241 submissions for necropsy examination or histopathology made to the California Animal Health and Food Safety Laboratory System between 1990 and 2007. The species affected most commonly were Amazon parrots (Amazona spp.)(n = 32; 26%) and grey-cheeked parakeets Brotogeris pyrrophterus (n = 23; 18.7%). The main gross findings on necropsy examination were enlarged and mottled pale livers and spleens and thickening of the small intestinal wall with numerous pale miliary nodules on the mucosa. Microscopical examination revealed infiltration of foamy macrophages and giant cells containing acid-fast bacteria in various organs. The gene encoding mycobacterial 65 kDa heat shock protein (hsp65) was amplified by nested polymerase chain reaction (PCR) from DNA extracted from 22 cases. The species of Mycobacterium involved was determined by analysis of restriction endonuclease patterns of the PCR products. Mycobacterium genavense was detected in 19 cases and Mycobacterium avium in two cases. One parrotlet (Touit spp.) had a mixed infection of both species of mycobacteria. It is concluded that M. genavense is the primary cause of mycobacteriosis in psittacine birds and the potential for zoonotic disease should be considered, especially for immunocompromised owners.

A 19S proteasomal subunit cooperates with an ERK MAPK-regulated degron to regulate accumulation of Fra-1 in tumour cells.

Fos-related antigen-1 (Fra-1) is a member of the Activator Protein-1 (AP-1) transcription factor superfamily that is overexpressed in a variety of cancers, including colon, breast, lung, bladder and brain. High Fra-1 levels are associated with enhanced cell proliferation, survival, migration and invasion. Despite its frequent overexpression, the molecular mechanisms that regulate the accumulation of Fra-1 proteins in tumour cells are not well understood. Here, we show that turnover of Fra-1, which does not require ubiquitylation, is cooperatively regulated by two distinct mechanisms-association with the 19S proteasomal subunit, TBP-1, and by a C-terminal degron, which acts independently of TBP-1, but is regulated by RAS-ERK (extracellular signal-regulated kinase) signalling. TBP-1 depletion stabilized Fra-1 and further increased its levels in tumour cells expressing RAS-ERK pathway oncogenes. These effects correlated with increased AP-1 transcriptional activity. We suggest that during Fra-1 degradation, association with TBP-1 provides a mechanism for ubiquitin-independent proteasomal recognition, while the C terminus of the protein regulates its subsequent proteolytic processing.

Fra-1 controls motility of bladder cancer cells via transcriptional upregulation of the receptor tyrosine kinase AXL.

Fos-related antigen 1 (Fra-1) is a Fos family member overexpressed in several types of human cancers. Here, we report that Fra-1 is highly expressed in the muscle-invasive form of the carcinoma of the bladder (80%) and to a lesser extent in superficial bladder cancer (42%). We demonstrate that in this type of cancer Fra-1 is regulated via a C-terminal instability signal and C-terminal phosphorylation. We show that manipulation of Fra-1 expression levels in bladder cancer cell lines affects cell morphology, motility and proliferation. The gene coding for AXL tyrosine kinase is directly upregulated by Fra-1 in bladder cancer and in other cell lines. Importantly, our data demonstrate that AXL mediates the effect of Fra-1 on tumour cell motility but not on cell proliferation. We suggest that AXL may represent an attractive therapeutic target in cancers expressing high Fra-1 levels.

Snail is a repressor of RKIP transcription in metastatic prostate cancer cells.

Diminished expression of the metastasis suppressor protein RKIP was previously reported in a number of cancers. The underlying mechanism remains unknown. Here, we show that the expression of RKIP negatively correlates with that of Snail zinc-transcriptional repressor, a key modulator of normal and neoplastic epithelial-mesenchymal transition (EMT) program. With a combination of loss-of-function and gain-of-function approaches, we showed that Snail repressed the expression of RKIP in metastatic prostate cancer cell lines. The effect of Snail on RKIP was on the level of transcriptional initiation and mediated by a proximal E-box on the RKIP promoter. Our results therefore suggest that RKIP is a novel component of the Snail transcriptional regulatory network important for the progression and metastasis of cancer.

MAP kinase signalling pathways in cancer.

Cancer can be perceived as a disease of communication between and within cells. The aberrations are pleiotropic, but mitogen-activated protein kinase (MAPK) pathways feature prominently. Here, we discuss recent findings and hypotheses on the role of MAPK pathways in cancer. Cancerous mutations in MAPK pathways are frequently mostly affecting Ras and B-Raf in the extracellular signal-regulated kinase pathway. Stress-activated pathways, such as Jun N-terminal kinase and p38, largely seem to counteract malignant transformation. The balance and integration between these signals may widely vary in different tumours, but are important for the outcome and the sensitivity to drug therapy.

High mortality in egg layers as a result of necrotic enteritis.

A new facility was designed to hold 1.8 million birds in 10 houses; chickens were placed in five of the houses, and the remaining five houses were under construction when this outbreak occurred. An increase in mortality was reported in five houses; however, mortality in house 7 was quite high. Well-fleshed birds were suddenly found dead without a significant drop in egg production. The middle and distal intestines were distended with gas, congested, thin walled, atonic, and bluish or pale in color with sloughed mucosa in some places. Necrotic enteritis was diagnosed as the cause of increased mortality. The ingesta in the crop occasionally contained flies. The 4-wk mortality in house 7 was 6.55% with a loss of 10,898 chickens. The 4-wk mortality rate in the other houses ranged from 0.54% to 1.98%. The houses affected with necrotic enteritis were treated for coccidiosis with amprolium because low numbers of the oocysts were present in the intestinal specimens of some of the chickens. Household bleach was added to the water at a dilution of one part bleach to 1040 parts water to control bacterial contamination. The fly (Musca domestica) population was out of control. Clostridium perfringens was isolated from the alcohol-washed macerated flies caught from houses 4 and 7. Dead flies were often seen in the feed troughs. The chickens may possibly have had C. perfringens infection as a result of consumption of dead flies or their secretions/excretions. The alcohol-washed, macerated, clarified fly extract from the affected houses caused death in 11 inoculated mice and paralysis in one mouse. Similarly, illness and mortality were present in four mice inoculated with clarified intestinal contents. The bacterium isolated on anaerobic culture was identified as C. perfringens by polymerase chain reaction. The disease was brought under control after straw was added and mixed in with the litter. As a result, the litter temperature increased, causing a decrease in the fly population. This study suggests that flies in the poultry houses acted as mechanical transmitters of C. perfringens and that the development of necrotic enteritis was by ingestion of bacteria present in the flies and their secretions/excretions.

Detection of avian polyomavirus infection by polymerase chain reaction using formalin-fixed, paraffin-embedded tissues.

Avian polyomavirus infection in psittacines was diagnosed in tissues by the use of polymerase chain reaction (PCR) test. The tissues used in the procedure were either formalin-fixed tissues embedded in paraffin blocks or fresh tissues (heart, liver, and spleen) collected from the psittacines during necropsy. DNA was extracted from these tissues and was tested with the published primers for avian polyomavirus VP1 gene in the PCR that yielded an amplicon of 550 base pair size, which was then visualized by electrophoresis. The amplicon size was consistent with avian polyomavirus. The PCR test was found to be an effective method for identifying avian polyomavirus infection in both formalin-fixed, paraffin-embedded and fresh tissues from psittacine birds of different age groups.

Diagnosis and management of gastro-oesophageal reflux in preterm infants in neonatal intensive care units.

AIM: There is relatively little published information regarding gastro-oesophageal reflux (GOR) in preterm infants, therefore the aim of this study was to elucidate the incidence of GOR and management regimes employed for this condition in major neonatal intensive care units (NICUs). METHODS: A standard questionnaire was sent to consultants in 77 level II (or secondary) and III (or tertiary) NICUs. RESULTS: Seventy-eight percent of consultants responded. Of babies born in these units, 40% were less than 34 wk gestational age and the estimated incidence of GOR in this group was 22%. GOR was diagnosed on a clinical basis alone in 42% of units, 8% used clinical features and/or investigations, and 50% used clinical features plus investigations and/or therapeutic trials. Intra-oesophageal pH monitoring was available in 93% of units but used regularly in only 32% of suspected cases. Common treatment strategies for diagnosed GOR included non-drug options--body positioning (98%) and placement on a slope (96%); and drugs--H2-receptor antagonists (100%), feed thickeners (98%), antacids (96%), prokinetic agents (79%), proton-pump inhibitors (65%) and dopamine-receptor antagonists (53%). However, the frequency with which all of these treatments were used varied widely between units. Surgery was required in only 1% of cases. CONCLUSIONS: GOR is perceived to be a common condition in preterm infants but the lack of published evidence relating to the management of GOR in preterm infants is reflected in the wide variation in diagnostic and treatment strategies used in major NICUs. It is clear that randomized, controlled trials to evaluate appropriate and effective treatments are needed.

Massive acute haemolysis in neonates with glucose-6-phosphate dehydrogenase deficiency.

Three neonates with glucose-6-phosphate dehydrogenase (G6PD) deficiency are described. All three patients suffered an episode of massive acute haemolysis, in the absence of blood group incompatibilities, infection, or ingestion of oxidising agents known to trigger haemolysis. One patient died, but the other two survived after an exchange transfusion. This highlights that G6PD deficiency in the neonatal period may present with severe anaemia in association with hyperbilirubinaemia.

Detection of pigeon circovirus by polymerase chain reaction.

Pigeon circovirus was identified by polymerase chain reaction (PCR) in young pigeons belonging to 12 different lofts. Viral DNA was extracted from formalin-fed, paraffin-imbedded tissues containing primarily bursa and occasionally liver and spleen with a commercial kit. PCR primers were selected from a published sequence for columbid circovirus and evaluated in a PCR assay. The histopathologic examination of various tissues revealed basophilic globular intracytoplasmic inclusions in the mononuclear cells of the bursa of Fabricius and occasionally in the spleen characteristic for a circovirus. Transmission electron microscopy of a few bursas of Fabricius revealed virus particles measuring 18-21 nm. All the samples were negative by PCR for psittacine beak and feather disease (PBFD) virus and chicken infectious anemia virus. The primers for both pigeon circovirus and PBFD virus did not react in PCR with the chicken anemia virus DNA. Most of the circovirus-infected pigeons had concurrent infections of Escherichia coli, Salmonella, Pasteurella, Aspergillus, candidiasis, nematodiasis, or capillariasis.

Results of salmonella isolation from poultry products, poultry, poultry environment, and other characteristics.

Five hundred sixty-nine Salmonella were isolated out of 4745 samples from poultry products, poultry, and poultry environment in 1999 and 2000 from the Pacific northwest. These Salmonella were identified to their exact source, and some were serogrouped, serotyped, phage typed, and tested for antibiotic sensitivity. Food product samples tested included rinse water of spent hens and broilers and chicken ground meat. Poultry environment samples were hatchery fluff from the hatcheries where eggs of grandparent broiler breeders or parent broiler breeder eggs were hatched and drag swabs from poultry houses. Diagnostic samples were of liver or yolk sac contents collected at necropsy from the young chicks received in the laboratory. Of these samples tested, 569 were Salmonella positive (11.99%). Ninety-two Salmonella were serogrouped with polyvalent somatic antisera A-I and the polymerase chain reaction. Somatic serogroups B and C comprised 95.25% of all the Salmonella. Out of a total of 569 positive samples, 97 isolates of Salmonella were serotyped. A total of 16 serotypes and an unnamed Salmonella belonging to serogroup C1 were identified. The Salmonella serotypes were heidelberg (25.77%); kentucky (21.64%); montevideo (11.34%); hadar and enteritidis (5.15% each); infantis, typhimurium, ohio, and thompson (4.12% each); mbandaka and cerro (3.09% each); senftenberg (2.06%); berta, istanbul, indiana, and saintpaul (1.03% each); and an unnamed monomorphic Salmonella (2.06%). Ninety-two Salmonella were tested for drug sensitivity with nine different antimicrobials. All of the 92 Salmonella were resistant to erythromycin, lincomycin, and penicillin except one sample (S. berta), which was moderately sensitive to penicillin. All of the tested Salmonella were susceptible to sarafloxacin and ceftiofur. The percentages of Salmonella susceptible to sulfamethoxazole-trimethoprim, gentamicin, triple sulfa, and tetracycline were 97.83%, 92.39%, 86.96%, and 82.61%, respectively.

Raf kinase inhibitor protein interacts with NF-kappaB-inducing kinase and TAK1 and inhibits NF-kappaB activation.

The Raf kinase inhibitor protein (RKIP) acts as a negative regulator of the mitogen-activated protein (MAP) kinase (MAPK) cascade initiated by Raf-1. RKIP inhibits the phosphorylation of MAP/extracellular signal-regulated kinase 1 (MEK1) by Raf-1 by disrupting the interaction between these two kinases. We show here that RKIP also antagonizes the signal transduction pathways that mediate the activation of the transcription factor nuclear factor kappa B (NF-kappaB) in response to stimulation with tumor necrosis factor alpha (TNF-alpha) or interleukin 1 beta. Modulation of RKIP expression levels affected NF-kappaB signaling independent of the MAPK pathway. Genetic epistasis analysis involving the ectopic expression of kinases acting in the NF-kappaB pathway indicated that RKIP acts upstream of the kinase complex that mediates the phosphorylation and inactivation of the inhibitor of NF-kappaB (IkappaB). In vitro kinase assays showed that RKIP antagonizes the activation of the IkappaB kinase (IKK) activity elicited by TNF-alpha. RKIP physically interacted with four kinases of the NF-kappaB activation pathway, NF-kappaB-inducing kinase, transforming growth factor beta-activated kinase 1, IKKalpha, and IKKbeta. This mode of action bears striking similarities to the interactions of RKIP with Raf-1 and MEK1 in the MAPK pathway. Emerging data from diverse organisms suggest that RKIP and RKIP-related proteins represent a new and evolutionarily highly conserved family of protein kinase regulators. Since the MAPK and NF-kappaB pathways have physiologically distinct roles, the function of RKIP may be, in part, to coordinate the regulation of these pathways.

Pathogenicity of environmental origin Salmonellas in specific pathogen-free chicks.

Two hundred sixty 1-d-old specific pathogen-free (SPF), Single Comb White Leghorn chicks were used in this study to determine pathology caused by Salmonella enteritidis (SE) isolated from a poultry environment. The chicks were subdivided into 10 equal groups of 26 chicks each. Eight groups of chicks were inoculated individually with 0.5 mL of brain heart infusion broth culture containing 1 x 10(6) cfu of SE phage type (PT) -8 (1, 2, 3), SE PT5 A (1, 2), or SE PT4 (Ch-env-CA, chicken-CA, and human) by crop gavage. One group of 26 chicks were inoculated with 1 x 10(6) cfu of Salmonella pullorum per bird by crop gavage. Another group of 26 chicks were kept as an uninoculated control group. All the chicks were observed daily for clinical signs and mortality. Salmonella was reisolated from different organs at 7, 14, 21, and 28 postinoculation (DPI). All of the chicks were weighed individually at each interval. Two chicks at random from each group were euthanised and necropsied at each DPI for gross pathology. Selected tissues were examined for histopathological changes at 7 and 14 DPI. Dead chicks were examined for gross and histopathological lesions. Mortality rates were 30.7, 15.3, and 7.6% in the groups inoculated with S. pullorum, SE PT5A, and SE PT4 (chicken-CA), respectively. No mortality or clinical sign were observed in other treatment groups or in uninoculated control groups. Cecal pouches were found to be the ideal organ for reisolation of Salmlonella at acute or chronic infection compared with other organs. Mean body weights were reduced to 1.8 to 12.6% in inoculated groups compared with the uninoculated control group. The consistent gross and hispathological lesions were of peritonitis, perihepatitis, yolk sac infection, and enteritis. Subclinical Salmlonella infection identified in this study resulted in reduced body weights of inoculated birds compared with uninoculated controls.

Alcoholic myopathy: biochemical mechanisms.

Between one- and two-thirds of all alcohol abusers have impairment of muscle function that may be accompanied by biochemical lesions and/or the presence of a defined myopathy characterised by selective atrophy of Type II fibres. Perturbations in protein metabolism are central to the effects on muscle and account for the reductions in muscle mass and fibre diameter. Ethanol abuse is also associated with abnormalities in carbohydrate (as well as lipid) metabolism in skeletal muscle. Ethanol-mediated insulin resistance is allied with the inhibitory effects of ethanol on insulin-stimulated carbohydrate metabolism. It acutely impairs insulin-stimulated glucose and lipid metabolism, although it is not known whether it has an analogous effect on insulin-stimulated protein synthesis. In alcoholic cirrhosis, insulin resistance occurs with respect to carbohydrate metabolism, although the actions of insulin to suppress protein degradation and stimulate amino acid uptake are unimpaired. In acute alcohol-dosing studies defective rates of protein synthesis occur, particularly in Type II fibre-predominant muscles. The relative amounts of mRNA-encoding contractile proteins do not appear to be adversely affected by chronic alcohol feeding, although subtle changes in muscle protein isoforms may occur. There are also rapid and sustained reductions in total (largely ribosomal) RNA in chronic studies. Loss of RNA appears to be related to increases in the activities of specific muscle RNases in these long-term studies. However, in acute dosing studies (less than 1 day), the reductions in muscle protein synthesis are not due to overt loss of total RNA. These data implicate a role for translational modifications in the initial stages of the myopathy, although changes in transcription and/or protein degradation may also be superimposed. These events have important implications for whole-body metabolism.

Cerebellar defect associated with Schimke immuno-osseous dysplasia.

UNLABELLED: We report the finding of an absent cerebellar hemisphere and partial absence of the cerebellar vermis in a child with dysmorphic features, spondyloepiphyseal dysplasia, steroid resistant nephrotic syndrome secondary to focal segmental glomerulosclerosis and T-cell lymphopenia (Schimke immuno-osseous dysplasia). These findings have not, to our knowledge, been described before and are likely to represent the consequence of a vascular event either in-utero or in early infancy. CONCLUSION: Cerebral imaging should be performed early in the course of the disease and should be repeated if further neurological events develop.

Pathogenicity of different serogroups of avian salmonellae in specific-pathogen-free chickens.

The pathogenicity of one isolate of Salmonella typhimurium, four isolates of Salmonella heidelberg, three isolates of Salmonella kentucky, two isolates of Salmonella montevideo, one isolate of Salmonella hadar, and two isolates of Salmonella enteritidis (SE), one belonging to phage type (PT)13a and the other to PT34, was investigated in specific-pathogen-free chicks. Three hundred eighty-four chicks were separated into 16 equal groups of 24 chicks. Thirteen groups were inoculated individually with 0.5 ml of broth culture containing 1 x 10(7) colony-forming units (CFU) of either S. typhimurium (one source), S. heidelberg (four sources), S. montevideo (two sources), S. hadar (one source), S. kentucky (three sources), SE PT 13a (one source) or SE PT 34 (one source) by crop gavage. Two groups of 24 chicks were inoculated in the same way with 1 x 10(7) CFU of SE PT4 (chicken-CA) and Salmonella pullorum. Another group of 24 chicks was kept as an uninoculated control group. The chicks were observed daily for clinical signs and mortality. Isolation of salmonella was done from different organs at 7 and 28 days postinoculation (DPI). All the chicks were weighed individually at 7, 14, 21, and 28 DPI. Two chicks chosen at random from each group were euthanatized and necropsied at 7 and 14 DPI and all the remaining live chickens, at 28 DPI. Selected tissues were taken for histopathology at 7 and 14 DPI. Dead chicks were examined for gross lesions and tissues were collected for histopathology. Chicks inoculated with S. pullorum had the highest mortality (66.66%), followed by S. typhimurium (33.33%). Chicks inoculated with S. heidelberg (00-1105-2) and SE PT4 (chicken-CA) had 12.5% mortality and 8.3% mortality, respectively, with SE PT 13a. Ceca were 100% positive for salmonellae at acute or chronic infection compared with other organs. Mean body weight reduction ranged from 0.67% (inoculated with S. kentucky 00-926-2) to 33.23% (inoculated with S. typhimurium 00-372) in the inoculated groups at different weeks compared with uninoculated controls. Gross and microscopic lesions included peritonitis, perihepatitis, yolk sac infection, typhilitis, pneumonia, and enteritis in some groups, especially those inoculated with S. typhimurium, S. heidelberg (00-1 105-2), SE PT4 (chicken-CA), and S. pullorum.

Rehab rounds: overcoming barriers to individualized psychosocial rehabilitation in an acute treatment unit of a state hospital.

Psychiatric rehabilitation begins during the acute stages of a psychiatric disorder and continues throughout the person's lifetime, with the types of services flexibly keyed to the person's phase of illness, needs, and personal goals. During periods of relapse and exacerbation of symptoms, when hospitalization is often required, psychiatric rehabilitation should include the following five objectives: * Clarify how the person's own goals in life, such as a desire for more self-control, freedom of choice, privacy, and time with friends and family, can be served by inpatient treatment and symptom stabilization. * Educate the patient about the nature of his or her illness and how medications work to restore self-control. * Teach the patient about side effects and self-monitoring and negotiating about medication and its effects in a collaborative way with the psychiatrist and other members of the treatment team. * Connect with the family or other natural supports that the person has in the community. * Enable the patient to make appropriate aftercare plans for residential and continuing treatment needs after discharge. When rehabilitation is viewed from the vantage point of these objectives, the inextricable interweaving of "treatment" with "rehabilitation" becomes clear. Treatment and rehabilitation are two sides of the same. It is much easier to integrate psychiatric rehabilitation into more traditional methods of treatment than it is to reorganize a treatment program or facility so that it blends rehabilitation with prevailing treatment imperatives of pharmacotherapy, supervision, and security and safety. In previous Rehab Rounds columns, we have described examples of creative methods for bringing the principles and practices of psychiatric rehabilitation into the treatment milieu (1,2,3). Faced with regulatory criticism from governmental agencies, Dr. Dhillon and his colleagues at Eastern State Hospital in Williamsburg, Virginia, launched a vigorous initiative to bring psychiatric rehabilitation into the forefront of their clinical enterprise. To enable readers to learn from their successful experience and adapt some of the administrative and clinical procedures that worked in Virginia, Dr. Dhillon and Ms. Dollieslager describe the operational details of their odyssey. We believe that their effectiveness in changing a traditional institution can be duplicated in many other places-in units within general hospitals or other community-based settings-as well as in state psychiatric hospitals, where acute treatment has been limited to pharmacotherapy and recreational and diversional activities.