Biomedical subjects

D J Donoghue

Publications and source records attributed to D J Donoghue.

At least 19 recordsLinked to original sources

PubMed2006

Upregulation of oxidative burst and degranulation in chicken heterophils stimulated with probiotic bacteria.

The immune system of neonatal chicks is functionally immature during the first week of life. Researchers have previously demonstrated that the avian humoral response can be increased with probiotics. Although the humoral response provides the chick with an effective mechanism to combat pathogens, sufficient antibody titers are not attained until 7 to 10 d postinfection. However, the innate immune system (i.e., heterophils) can respond much more quickly to pathogens. The objective of this study was to determine whether probiotic bacteria can also upregulate heterophil function. Heterophils were isolated from the peripheral blood of neonatal chickens by using a discontinuous density gradient. Oxidative burst and degranulation are bactericidal mechanisms used by heterophils to kill pathogens and were used in this study as indicators of heterophil function. We found that each of the 10 "generally recognized as safe" probiotic isolates (designated G1 to G11) tested in vitro were capable of increasing (P < 0.05) heterophil oxidative burst and degranulation when compared with unstimulated controls. Bacillus subtilis (G3), Lactococcus lactis lactis (G6), and Lactobacillus acidophilus (G8) isolates were determined to elicit the greatest heterophil response in vitro and were subsequently fed to chicks. Phosphate-buffered saline or 1 of these 3 probiotic isolates (approximately 2.5 x 10(8) cfu/chick; 50 chicks/treatment) resuspended in PBS was administered by oral gavage on the day of hatch. Heterophils were isolated from chicks from each of these 4 treatment groups 24 h posttreatment. Significant increases in heterophil degranulation and oxidative burst were observed with the G3-, G6-, and G8-treated chicks when compared with heterophils isolated from birds with no probiotic treatment. These data suggest that probiotic bacteria can significantly improve heterophil oxidative burst and degranulation in broilers. To our knowledge, this is the first study demonstrating a relationship between probiotics and avian heterophil function.

Animals↗

PubMed2006

Effect of oral administration of bismuth compounds on Campylobacter colonization in broilers.

Bismuth compounds have been used since the 18th century to treat gastrointestinal ailments in man. Colloidal bismuth subcitrate (De-Nol) is currently used in combination with antibiotics to reduce enteric Helicobacter pylori colonization as a treatment of stomach ulcers. We investigated whether bismuth citrate or its parent compound, colloidal bismuth subcitrate, would reduce colonization of the closely related foodborne pathogen, Campylobacter jejuni in chickens. In 2 studies, birds were either fed 0, 50, or 200 ppm bismuth citrate or bismuth subcitrate (De-Nol) for 10 or 21 d and were orally challenged with 7 combined strains of C. jejuni (n = 6 birds/treatment). For both treatment groups, cecal Campylobacter colonization was reduced when birds were fed 200 ppm for 10 d but not 21 d. For the 50 ppm treatment group, only birds dosed with bismuth citrate for 21 d demonstrated any reduction in cecal Campylobacter concentrations when compared with controls. These data suggest that bismuth citrate and colloidal bismuth subcitrate may reduce cecal colonization by Campylobacter in broilers, but these effects are inconsistent.

Administration, Oral↗

PubMed2006

Efficacy of iron chelators on Campylobacter concentrations in turkey semen.

Campylobacter is a leading bacterial cause of human foodborne infections in the United States. Recent studies suggest that the organism is highly prevalent in poultry semen and may contribute to vertical transmission between the breeder hen and offspring. Because Campylobacter requires iron for its growth and survival, the objective of this study was to determine if the addition of natural and synthetic chelators such as ovotransferrin, desferrioxaime, EDTA, or 2,2'-dipyridyl could reduce or eliminate Campylobacter in turkey semen. In a preliminary study without semen, a commercial poultry semen extender was supplemented with various concentrations of ovotransferrin, desferrioxaime, EDTA, or 2,2'-dipyridyl and inoculated with an average of 10(8) cfu/mL of a wild-type Campylobacter coli turkey semen isolate. At 6 and 24 h of storage at 4 degrees C, a sample was taken from each treatment group and enumerated for Campylobacter. In all 3 trials, Campylobacter was undetectable (< 10(2)) in the commercial poultry semen extender supplemented with 20 mg/mL of 2,2'-dipyridyl. There were no differences observed in Campylobacter concentrations in the commercial poultry semen extender supplemented with ovotransferrin, desferrioxaime, or EDTA compared with unsupplemented controls. In a follow-up study, pooled semen samples were randomly collected from toms, diluted with a commercial poultry semen extender supplemented with 5, 10, or 20 mg/mL of 2,2'-dipyridyl and inoculated with an average of 10(8) cfu/mL of a wild-type C. coli turkey semen isolate. At 6 and 24 h of storage at 4 degrees C, samples were taken from each treatment group, enumerated for Campylobacter, and evaluated for sperm viability. In all 3 trials, supplementing the commercial poultry semen extender with 20 mg/mL of 2,2'-dipyryidyl significantly reduced (3 to 4 logs) Campylobacter concentrations when compared with the positive controls. Sperm viability was also reduced with this treatment, and, therefore, the use of 2,2'-dipyridyl may not be a practical treatment for reducing Campylobacter in poultry semen.

2,2'-Dipyridyl↗

PubMed2006

Bacteriocins reduce Campylobacter colonization and alter gut morphology in turkey poults.

Campylobacter is a leading cause of food-borne illness in the United States. Recent evidence has demonstrated that bacteriocins produced by Bacillus circulans and Paenibacillus polymyxa reduce cecal Campylobacter colonization in broiler chickens infected with Campylobacter jejuni. As Campylobacter coli is the most prevalent Campylobacter isolate recovered in turkeys, the objectives of the present study were to evaluate the efficacy of these bacteriocins against C. coli colonization and their influence on the gastrointestinal architecture of young turkeys. In 3 separate trials, a total of 135 day-of-hatch poults (n = 45/trial) were orally challenged on d 3 with approximately 10(6) cfu of a mixture of 3 C. coli isolates. Immediately before bacteriocin treatment (d 10), cecal Campylobacter concentrations averaged 1.1 x 10(7) cfu/ g of cecal contents (n = 15/trial). On d 10 to 12 posthatch, 2 bacteriocin treatment groups were given free access to feed supplemented with purified, microencapsulated bacteriocins, whereas the positive control treatment group had access to untreated feed (n = 10/treatment group per trial). At the end of the 3-d dosing period, ceca and duodenal loops were collected for analysis. In each of the 3 separate trials, treatment with bacteriocin eliminated detectable ceca Campylobacter concentrations (detection limit, 1 x 10(2) cfu/g of cecal contents) vs. controls (1.0 x 106 cfu of Campylobacter/g of cecal contents). Duodenum crypt depth and goblet cell numbers were also reduced in turkeys treated with either bacteriocin vs. controls (P < 0.05). The dynamic reduction in crypt depth and goblet cell density in turkeys dosed with bacteriocin may provide clues to how bacteriocins inhibit enteric Campylobacter.

Animal Feed↗

PubMed2005

Use of a specific bacteriophage treatment to reduce Salmonella in poultry products.

Bacteriophages represent a group of viruses that specifically infect and replicate in bacteria and could potentially be used to reduce recovery of Salmonella from poultry carcasses. Bacteriophages were isolated from municipal wastewater in the presence of Salmonella enteritidis phage type 13A (SE). In the first 2 experiments, commercially processed broiler carcass rinse water was pooled and divided. The addition of 10(10) pfu/mL of a single bacteriophage (PHL 4) with selected concentrations of SE reduced (P < 0.05) frequency of SE recovered as compared with the control rinse water sample. In experiments 3 and 4, broiler carcasses were intentionally inoculated with SE, sprayed with selected concentrations of PHL 4, and rinsed for SE enrichment and isolation. Application of 5.5 mL of 10(8) or 10(10) pfu/mL of PHL 4 reduced (P < 0.05) the frequency of SE recovery as compared with controls. In experiments 5 and 6, commercially processed turkeys were rinsed with water containing 72 wild-type bacteriophages isolated against SE, which were amplified in SE, or the Salmonella isolated antemortem from drag swabs from the flock selected for in-plant treatment, or a combination of bacteriophages amplified by each bacterial host. All bacteriophage treatments reduced (P < 0.05) frequency of Salmonella recovery as compared with controls. Sufficient concentrations of an appropriate bacteriophage, or a bacteriophage mixture, can significantly reduce recoverable Salmonella from carcass rinses.

Animals↗

PubMed2005

Campylobacter susceptibility to ciprofloxacin and corresponding fluoroquinolone concentrations within the gastrointestinal tracts of chickens.

AIMS: This study evaluated the relationship between Campylobacter susceptibility and enteric fluoroquinolone concentrations in chickens treated with different doses of enrofloxacin. METHODS AND RESULTS: All chickens were challenged with seven fluoroquinolone sensitive Campylobacter jejuni (6.6 x 10(6) CFU per bird) at 2 weeks posthatch. At 26 days of age chickens were treated with 0 (n = 29 birds), 25 mg ml(-1) enrofloxacin (Baytril, Bayer Corp., Shawnee Mission, KS, USA) for 3 days (n = 45 birds) or 50 mg ml(-1) enrofloxacin for 7 days (n = 65 birds) in the drinking water. The crop, upper ileum, lower ileum, ceca and colon contents were collected from both enrofloxacin treatment groups (n = 5 birds per day per treatment group) and nonmedicated controls. The minimum inhibitory concentration (MIC) of ciprofloxacin for Campylobacter increased for isolates from both treatment groups within the first day of dosing and the daily average ranged from 1.4 to 6.5 microg ml(-1) throughout the study. Although enteric fluoroquinolone concentrations were higher (P < 0.05) in birds dosed with 50 mg ml(-1)vs 25 mg ml(-1) enrofloxacin, there were no differences between the isolates collected from these groups for MIC values. CONCLUSION: These data indicate, for the doses used, differences in gut fluoroquinolone concentrations do not produce isolates of Campylobacter with differing susceptibility to ciprofloxacin. SIGNIFICANCE AND IMPACT OF THE STUDY: Using the manufacturers lowest, shortest duration dose vs the highest, longest duration dose of enrofloxacin did not change Campylobacter susceptibility to ciprofloxacin. However, ciprofloxacin MIC values for Campylobacter determined in this study were lower than previously reported.

Animals↗

PubMed2004

Detection of Campylobacter or Salmonella in turkey semen and the ability of poultry semen extenders to reduce their concentrations.

Campylobacter and Salmonella are the most commonly reported pathogens causing foodborne illness in the United States. In turkeys, the potential that semen used for artificial insemination is contaminated with these foodborne pathogens has not been investigated. Because semen on turkey farms is pooled and then used to inseminate multiple hens, contaminated semen could easily spread these bacteria throughout entire flocks via artificial insemination. The objectives of this study were to 1) determine if semen from commercial turkey farms contained these foodborne pathogens and 2) if present, evaluate the efficacy of semen extenders to reduce or eliminate Campylobacter and Salmonella from semen. Semen was collected from randomized pools of ejaculates from 10 to 30 toms per farm from 6 flocks over a 7-wk period and, on occasion, was found to contain Campylobacter, Salmonella, or both. To evaluate the efficacy of semen extenders to reduce or eliminate pathogens, pooled ejaculates were challenged with Campylobacter or Salmonella and treated with commercial poultry extenders containing various concentrations of antibiotics or an antibiotic combination previously demonstrated to remove Campylobacter from mammalian semen. Results demonstrate that commercial turkey semen may contain Campylobacter or Salmonella, and the semen extenders tested either did not reduce the bacteria or reduced but did not eliminate these bacteria from semen. We concluded that semen may be a potential vehicle for Campylobacter transfer to hens, and, if this is true, development of a method for eliminating pathogens in semen before insemination could reduce the risk of colonization.

Animals↗

PubMed2004

Effects of aeration and storage temperature on Campylobacter concentrations in poultry semen.

Campylobacter is one of the most commonly reported bacterial causes of human foodborne infections in the United States. Recent evidence has demonstrated that Campylobacter is present in poultry semen and may contribute to the vertical transmission between the breeder hen and offspring. As Campylobacter is considered sensitive to oxygen and cold temperature, the objective of this study was to determine if aeration and storage temperature could reduce or eliminate Campylobacter in poultry semen. In 4 separate trials, pooled semen samples were collected from roosters or toms, diluted with a commercial poultry semen extender, and inoculated with an average of 10(7) cells/mL of a wild-type C. jejuni or C. coli semen isolate. Pooled ejaculates were then divided into 3 aeration treatments: Control (no aeration), air, or oxygen (gently bubbled for 20 min with atmospheric air or oxygen, respectively). Immediately after aeration, pooled semen samples were further divided to 3 test storage temperatures: 4, 23, or 42 degrees C. At 0, 2, 6, and 24 h of storage, samples were enumerated for Campylobacter. Aeration of the semen alone, or aeration with reduced temperatures (4 or 23 degrees C), did not reduce the amount of Campylobacter compared with the controls. Campylobacter concentrations were, however, reduced when stored at 42 degrees C for 24 h. This effect is associated with reduced sperm viability and is therefore not a practical treatment of reducing Campylobacter in semen. It appears alternative methods will be needed to eliminate Campylobacter from poultry semen.

Animals↗

PubMed2004

Comparison of a bioassay and a liquid chromatography-fluorescence-mass spectrometry(n) method for the detection of incurred enrofloxacin residues in chicken tissues.

Regulatory monitoring for most antibiotic residues in edible poultry tissues is often accomplished with accurate, although expensive and technically demanding, chemical analytical techniques. The purpose of this study is to determine if a simple, inexpensive bioassay could detect fluoroquinolone (FQ) residues in chicken muscle above the FDA established tolerance (300 ppb) comparable to a liquid chromatography-fluorescencemass spectrometry(n) method. To produce incurred enrofloxacin (ENRO) tissues (where ENRO is incorporated into complex tissue matrices) for the method comparison, 40-d-old broilers (mixed sex) were orally dosed through drinking water for 3 d at the FDA-approved dose of ENRO (50 ppm). At the end of each day of the 3-d dosing period and for 3 d postdosing, birds were sacrificed and breast and thigh muscle collected and analyzed. Both methods were able to detect ENRO at and below the tolerance level in the muscle, with limits of detection of 26 ppb (bioassay), 0.1 ppb for ENRO, and 0.5 ppb for the ENRO metabolite, ciprofloxacin (liquid chromatography-fluorescence-mass spectrometry(n)). All samples that had violative levels of antibiotic were detected by the bioassay. These results support the use of this bioassay as a screening method for examining large numbers of samples for regulatory monitoring. Positive samples should then be examined by a more extensive method, such as liquid chromatography-fluorescence-mass spectrometry(n), to provide confirmation of the analyte.

Animals↗

PubMed2004

Isolation and prevalence of Campylobacter in the reproductive tracts and semen of commercial turkeys.

Campylobacter is one of the most commonly reported bacterial causes of human foodborne infections in the United States, and epidemiologic evidence indicates that a significant proportion of human infections result from the improper preparation of poultry products. Campylobacter frequently colonizes the avian intestinal tract, but recent research indicates that this organism can also colonize the avian reproductive tract and possibly contaminate eggs and subsequent offspring. The present studies were undertaken to determine the prevalence of Campylobacter in the reproductive systems of commercial turkeys. In the first study, pooled semen samples from seven commercial turkey farms were randomly collected by abdominal massage over a period of 13 wk. The pooled semen samples were serially diluted, and 0.1 ml of each dilution was plated on Campy-Line agar and incubated at 42 C for 48 hr in a microaerophilic environment for enumeration of Campylobacter. Campylobacter was isolated from 57 of the 59 pooled semen samples, and levels ranged from below the limit of detection (<10(1)) to 1.6 x 10(6) cfu/ml of semen. In the second study, the reproductive tracts of 11 hens and 17 toms were aseptically excised, and the segments (female: vagina, shell gland, isthmus, magnum, and infundibulum; male: ductus deferens and testes) were swabbed with a dry cotton sterile swab. The swabs were incubated for 24 hr in Campylobacter enrichment broth, and 0.1 ml of the enriched sample solution was streaked onto Campy-Line agar plates and incubated at 42 C for 48 hr in a microaerophilic environment. Of the 11 hens sampled, Campylobacter was isolated from the vagina (10/11), the shell gland (7/11), the isthmus (8/11), the magnum (6/11), and the infundibulum (4/11). Of the 17 toms sampled, Campylobacter was isolated from the ductus deferens (8/17) and the testes (2/17). Campylobacter is present in the reproductive tracts and semen of commercial turkeys and may lead to vertical transmission of Campylobacter from the hen to the chick.

Animals↗

PubMed2003

Field testing the influence of sperm competition based on sperm mobility in breeder turkey toms.

1. Commercial reproduction of turkeys relies on pooling of semen from multiple males for inseminations. Understanding how sperm characteristics influence paternity under commercial breeding conditions is important to improving production efficiency. 2. The objective of this study was to evaluate progeny production of individual toms following commercial practices of pooling semen to determine if sperm mobility influences progeny production in field conditions. 3. A total of 104 toms were evaluated for sperm mobility. A subset of 10 toms were housed together and semen was collected, pooled and used to inseminate hens (n = 28). Hens were inseminated at 30 weeks of age and weekly thereafter. 4. Ejaculates from each tom were evaluated on two separate days for sperm mobility. Semen from each tom was diluted and layered upon 6% (wt/vol) Accudenz solution. The sperm suspension was incubated at 41 degrees C for 5 min and absorbance was measured with a spectrophotometer. 5. Toms were ranked by absorbance and categorised as high or low if mobility score was +/- 1 SD from the flock mean (average). 6. For parentage determination, DNA was extracted from tom, hen and poult blood. Poult parentage (n = 276) was determined at one day of age or at 14 weeks by analysis of marker genotypes that were generated by polymerase chain reaction (PCR) amplification of genomic DNA with selected microsatellite markers. 7. Sperm mobility differed across males with absorbance values ranging from 0.147 to 0.366. 8. Findings demonstrate differences in poult production among individual toms when semen from multiple males was pooled and inseminated. Toms classified as high, average and low produced 55, 41 and 4% of the offspring, respectively. 9. It appears that sperm mobility is a trait that influences sperm competition among toms under field conditions where sperm numbers inseminated from individual toms are not controlled or constant and that toms with low sperm mobility produce few offspring.

Animals↗

PubMed2003

Approach for selection of individual enteric bacteria for competitive exclusion in turkey poults.

The intentional early colonization of the intestinal tract with beneficial microflora, known as competitive exclusion, has been shown to successfully protect poultry from selected enteric pathogens. Although effective cultures have been produced and are available, an inexpensive, air-tolerant, and completely defined culture is needed. Presently, we developed an in vitro competition assay to select for individual facultative anaerobes of poultry enteric origin that could exclude Salmonella. Using this assay, 24 isolates were selected and stored individually. These 24 isolates were amplified in batch culture (tryptic soy broth, 4 h at 40 degrees C) and administered at final dilutions of 10, 100, or 1,000 cfu to day-of-hatch poults. Forty-eight hours later, poults were challenged with 100 to 1,000 cfu antibiotic-resistance-marked Salmonella enteritidis PT 13A by oral gavage. Five days later, all poults were killed, and cecal tonsils were aseptically removed for tetrathionate enrichment (24 h at 37 degrees C) followed by selective plating with marker antibiotics. Selected lactose-negative, antibiotic-resistant colonies typical of Salmonella were further confirmed by serogrouping. Treatment-related protection ranged from 0 to 100% in three experiments. Greatest protection was related to the lowest concentrations of the protective microflora in each experiment. These data suggest that effective combinations of competitive enteric microflora can be identified by appropriate in vitro selection methods.

Animal Husbandry↗

PubMed2001

Patched1 interacts with cyclin B1 to regulate cell cycle progression.

The initiation of mitosis requires the activation of M-phase promoting factor (MPF). MPF activation and its subcellular localization are dependent on the phosphorylation state of its components, cdc2 and cyclin B1. In a two-hybrid screen using a bait protein to mimic phosphorylated cyclin B1, we identified a novel interaction between cyclin B1 and patched1 (ptc1), a tumor suppressor associated with basal cell carcinoma (BCC). Ptc1 interacted specifically with constitutively phosphorylated cyclin B1 derivatives and was able to alter their normal subcellular localization. Furthermore, addition of the ptc1 ligand, sonic hedgehog (shh), disrupts this interaction and allows cyclin B1 to localize to the nucleus. Expression of ptc1 in 293T cells was inhibitory to cell proliferation; this inhibition could be relieved by coexpression of a cyclin B1 derivative that constitutively localizes to the nucleus and that could not interact with ptc1 due to phosphorylation-site mutations to ALA: In addition, we demonstrate that endogenous ptc1 and endogenous cyclin B1 interact in vivo. The findings reported here demonstrate that ptc1 participates in determining the subcellular localization of cyclin B1 and suggest a link between the tumor suppressor activity of ptc1 and the regulation of cell division. Thus, we propose that ptc1 participates in a G(2)/M checkpoint by regulating the localization of MPF.

Animals↗

PubMed2001

Analysis of pesticide residues in eggs by direct sample introduction/gas chromatography/tandem mass spectrometry.

Direct sample introduction (DSI) or "dirty sample injection" is a rapid, rugged, and inexpensive approach to large volume injection in gas chromatography (GC) for semivolatile analytes such as pesticides. DSI of complex samples such as eggs requires a very selective detection technique, such as tandem mass spectrometry (MS-MS), to determine the analytes among the many semivolatile matrix components that also appear. In DSI, the nonvolatile matrix components that normally would contaminate the GC system in traditional injection methods remain in a disposable microvial, which is removed after every injection. For example, 3 microg of nonvolatile residue typically remained in the microvial after an injection of egg extract using the DSI method. This analytical procedure involves the following: (i) weighing 10 g of egg in a centrifuge tube and adding 2 g of NaCl and 19.3 mL of acetonitrile (MeCN); (ii) blending for 1 min using a probe blender; (iii) centrifuging for 10 min; and (iv) analyzing 10 microL (5 mg of egg equivalent) of the extract using DSI/GC/MS-MS. No sample cleanup or solvent evaporation steps were required to achieve quantitative and confirmatory results with <10 ng/g detection limits for 25 of 43 tested pesticides from several chemical classes. The remaining pesticides gave higher detection limits due to poor fragmentation characteristics in electron impact ionization and/or degradation. Analysis of eggs incurred with chlorpyrifos-methyl showed a similar trend in the results as a more traditional approach.

Desiccation↗

PubMed2001

Identification of tyrosine residues in constitutively activated fibroblast growth factor receptor 3 involved in mitogenesis, Stat activation, and phosphatidylinositol 3-kinase activation.

Fibroblast growth factor receptor 3 (FGFR3) mutations are frequently involved in human developmental disorders and cancer. Activation of FGFR3, through mutation or ligand stimulation, results in autophosphorylation of multiple tyrosine residues within the intracellular domain. To assess the importance of the six conserved tyrosine residues within the intracellular domain of FGFR3 for signaling, derivatives were constructed containing an N-terminal myristylation signal for plasma membrane localization and a point mutation (K650E) that confers constitutive kinase activation. A derivative containing all conserved tyrosine residues stimulates cellular transformation and activation of several FGFR3 signaling pathways. Substitution of all nonactivation loop tyrosine residues with phenylalanine rendered this FGFR3 construct inactive, despite the presence of the activating K650E mutation. Addition of a single tyrosine residue, Y724, restored its ability to stimulate cellular transformation, phosphatidylinositol 3-kinase activation, and phosphorylation of Shp2, MAPK, Stat1, and Stat3. These results demonstrate a critical role for Y724 in the activation of multiple signaling pathways by constitutively activated mutants of FGFR3.

3T3 Cells↗

PubMed2000

Distinct missense mutations of the FGFR3 lys650 codon modulate receptor kinase activation and the severity of the skeletal dysplasia phenotype.

The fibroblast growth factor-receptor 3 (FGFR3) Lys650 codon is located within a critical region of the tyrosine kinase-domain activation loop. Two missense mutations in this codon are known to result in strong constitutive activation of the FGFR3 tyrosine kinase and cause three different skeletal dysplasia syndromes-thanatophoric dysplasia type II (TD2) (A1948G [Lys650Glu]) and SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans) syndrome and thanatophoric dysplasia type I (TD1) (both due to A1949T [Lys650Met]). Other mutations within the FGFR3 tyrosine kinase domain (e.g., C1620A or C1620G [both resulting in Asn540Lys]) are known to cause hypochondroplasia, a relatively common but milder skeletal dysplasia. In 90 individuals with suspected clinical diagnoses of hypochondroplasia who do not have Asn540Lys mutations, we screened for mutations, in FGFR3 exon 15, that would disrupt a unique BbsI restriction site that includes the Lys650 codon. We report here the discovery of three novel mutations (G1950T and G1950C [both resulting in Lys650Asn] and A1948C [Lys650Gln]) occurring in six individuals from five families. Several physical and radiological features of these individuals were significantly milder than those in individuals with the Asn540Lys mutations. The Lys650Asn/Gln mutations result in constitutive activation of the FGFR3 tyrosine kinase but to a lesser degree than that observed with the Lys540Glu and Lys650Met mutations. These results demonstrate that different amino acid substitutions at the FGFR3 Lys650 codon can result in several different skeletal dysplasia phenotypes.

Adolescent↗

PubMed2000

Transformation and Stat activation by derivatives of FGFR1, FGFR3, and FGFR4.

The fibroblast growth factor receptor (FGFR) family members mediate a number of important cellular processes, and are mutated or overexpressed in several forms of human cancer. Mutation of Lys650-->Glu in the activation loop of the FGFR3 kinase domain causes the lethal human skeletal disorder thanatophoric dysplasia type II (TDII) and is also found in patients with multiple myeloma, bladder and cervical carcinomas. This mutation leads to constitutive activation of FGFR3. To compare the signaling activity of FGFR family members, this activating mutation was generated in FGFR1, FGFR3, and FGFR4. We show that the kinase domains of FGFR1, FGFR3, and FGFR4 containing the activation loop mutation, when targeted to the plasma membrane by a myristylation signal, can transform NIH3T3 cells and induce neurite outgrowth in PC12 cells. Phosphorylation of Shp2, PLC-gamma, and MAPK was also stimulated by all three 'TDII-like' FGFR derivatives. Additionally, activation of Stat1 and Stat3 was observed in cells expressing the activated FGFR derivatives. Finally, we demonstrate that FGFR1, FGFR3, and FGFR4 derivatives can stimulate PI-3 kinase activity. Our comparison of these activated receptor derivatives reveals a significant overlap in the panel of effector proteins used to mediate downstream signals. This also represents the first demonstration that activation of FGFR4, in addition to FGFR1 and FGFR3, can induce cellular transformation. Moreover, our results suggest that Stat activation by FGFRs is important in their ability to act as oncogenes.

3T3 Cells↗

PubMed2000

Activation of H-ras61L-specific signaling pathways does not require posttranslational processing of H-ras.

We have previously demonstrated that H-ras61L retained transforming activity when lacking C-terminal lipid modifications, provided that plasma membrane localization was restored by an N-terminal transmembrane domain. Since several ras-activated pathways contribute to the transformed phenotype, we utilized a novel set of transmembrane domain-anchored H-ras derivatives to examine if lipids are required for activation of any specific signaling pathways. We demonstrate here that H-ras61L-induced activation of the Raf/MEK/MAPK pathway, including recruitment of Raf to the plasma membrane and activation of Raf and MAPK, does not require C-terminal processing of H-ras61L. Biochemical fractionation experiments confirm the localization of TM-ras derivatives to the plasma membrane, as well as the ras-mediated recruitment of c-Raf-1. Changes in the actin cytoskeleton, controlled by H-ras61L-mediated activation of the Rac/ Rho pathway, as well as PI 3-kinase activation, can also occur in the absence of C-terminal lipid modifications. Finally, downstream events, such as the induction of the immediate-early gene c-fos or neurite outgrowth in PC12 cells, are stimulated by the expression of plasma membrane-anchored, nonlipidated H-ras6lL. These results demonstrate that H-ras can be functionally targeted to the plasma membrane using a transmembrane domain sequence and that several signal transduction pathways downstream of H-ras can be activated without the presence of normal lipid modifications.

Animals↗