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Biomedical subjects

S E Craven

Publications and source records attributed to S E Craven.

At least 19 recordsLinked to original sources
PubMed2006

Molecular subtyping of poultry-associated type A Clostridium perfringens isolates by repetitive-element PCR.

Clostridium perfringens strains (type A) isolated from an integrated poultry operation were subtyped using repetitive-element PCR with Dt primers. Isolates were obtained from fecal, egg shell, fluff, and carcass rinse samples as part of a previously reported temporally linked epidemiological survey. A total of 48 isolates of C. perfringens were obtained from different stages of the broiler chicken production chain from two separate breeder farms that supplied a single hatchery that in turn provided chicks to a single grow-out farm whose flocks were processed at a single plant. All 48 isolates were typeable (100% typeability) by repetitive-element PCR with Dt primers. This subtyping method was highly reproducible and discriminatory. By repetitive-element PCR with Dt primers, isolates were classified into four major branches with 12 subgroups or clades. The Simpson's index of discrimination was calculated to be 0.96 for groupings of >95% correlation. Toxin gene profiles of the isolates indicated that all of the isolates were C. perfringens alpha-toxin gene positive and 46 of 48 isolates were beta2-toxin gene positive. All strains were negative for beta- and epsilon-toxin genes. Repetitive sequence-based PCR was found to be a technically practical and reproducible means of subtyping C. perfringens libraries from specific epidemiological or production environment settings.

Animals↗
PubMed2002

Serotype tracking of Salmonella through integrated broiler chicken operations.

The widespread presence of Salmonella in all phases of broiler chicken production and processing is well documented. However, little information is available to indicate the identity and movement of specific serotypes of Salmonella through the different phases of an integrated operation. In this study, samples were collected from the breeder farm, from the hatchery, from the previous grow-out flock, from the flock during grow-out, and from carcasses after processing. Salmonella were recovered from 6, 98, 24, 60, and 7% of the samples, respectively, in the first trial and from 7, 98, 26, 22, and 36% of the samples, respectively, in the second trial. Seven different serotypes were identified in the first trial, and 12 different serotypes were identified in the second trial. For both trials there was poor correlation between the serotypes found in the breeder farms and those found in the hatchery. This finding and the fact that similar serotypes were found in the hatchery in both trials suggests that there was an endemic population of Salmonella in the hatchery. An association between the serotypes found in the hatchery and those found on the final processed carcasses was observed in both trials. This study confirms that a successful intervention program for broiler production operations must be multifaceted, with one component being disinfection in the hatchery.

Animals↗
PubMed2002

Serotyping and ribotyping of Salmonella using restriction enzyme PvuII.

The subtyping and identification of bacterial pathogens throughout food processing and production chains is useful to the new hazard analysis critical control point-based food safety plans. Traditional manual serotyping remains the primary means of subtyping Salmonella isolates. Molecular biology techniques, however, offer the promise of more rapid and sensitive subtyping of Salmonella. This study evaluates the potential of restriction enzyme PvuII, followed by probing with the rRNA operon from Escherichia coli, to generate serotype-specific DNA fingerprints. A total of 32 identified serotypes were found with an overall agreement in 208 of the 259 (80%) isolates tested between U.S. Department of Agriculture serotype identification and riboprint serotype identification. Many of the isolates that did not correlate were serotype identified as Salmonella Montevideo, which indicates that for this serotype, there are multiple ribotypes. When Salmonella Montevideo isolates were not included, the ribotype identification agreed with serotyping in 207 of the 231 (90%) isolates. The primary outcome of any ribotyping procedure is to give distinct ribotype patterns. This extensive poultry epidemiological study demonstrates that, in addition to ribotype patterns, the identification of isolates to known serotypes provides the investigator with additional information that can be more useful than traditional epidemiology and isolate identification studies.

Animals↗
PubMed2001

Polarized targeting of peripheral membrane proteins in neurons.

Differential targeting of neuronal proteins to axons and dendrites is essential for directional information flow within the brain, however, little is known about this protein-sorting process. Here, we investigate polarized targeting of lipid-anchored peripheral membrane proteins, postsynaptic density-95 (PSD-95) and growth-associated protein-43 (GAP-43). Whereas the N-terminal palmitoylated motif of PSD-95 is necessary but not sufficient for sorting to dendrites, the palmitoylation motif of GAP-43 is sufficient for axonal targeting and can redirect a PSD-95 chimera to axons. Systematic mutagenesis of the GAP-43 and PSD-95 palmitoylation motifs indicates that the spacing of the palmitoylated cysteines and the presence of nearby basic amino acids determine polarized targeting by these two motifs. Similarly, the axonal protein paralemmin contains a C-terminal palmitoylated domain, which resembles that of GAP-43 and also mediates axonal targeting. These axonally targeted palmitoylation motifs also mediate targeting to detergent-insoluble glycolipid-enriched complexes in heterologous cells, suggesting a possible role for specialized lipid domains in axonal sorting of peripheral membrane proteins.

Amino Acid Motifs↗
PubMed2001

Sources and movement of Salmonella through integrated poultry operations: a multistate epidemiological investigation.

The prevalence of Salmonella from numerous sources in 32 integrated broiler operations of high- and low-performing broiler houses was characterized from four states across four seasons. Previous studies of Salmonella in broilers have been limited in scope, offering only a snapshot of pathogen prevalence as seen on a small number of individual farms. Twenty-six different sample types were collected from the hatchery to the end of processing, and Salmonella was found in all sample types. A total of 10,740 samples were analyzed for Salmonella, and 973 (9.1%) of these samples, including 49 of 798 (6.1%) carcass rinse samples, were Salmonella positive. Hatchery transport pads (389 of 765, 50.8%), flies (28 of 150, 18.7%), drag swabs (57 of 402, 14.2%), and boot swabs (20 of 167, 12%) were samples from which Salmonella was most frequently isolated. Thirty-six different serotypes were identified, and the most frequently encountered serotypes were Salmonella Senftenberg, Salmonella Thompson, and Salmonella Montevideo. Determining critical contaminating sources and following the movement of Salmonella through integrated poultry operations will help researchers and the industry develop practical intervention strategies.

Animals↗
PubMed2001

Distribution of Campylobacter spp. in selected U.S. poultry production and processing operations.

A study was conducted of 32 broiler flocks on eight different farms, belonging to four major U.S. producers. The farms were studied over I complete calendar year. Overall, 28 (87.5%) of the flocks became Campylobacter positive, and only four (12.5%) remained negative throughout the 6- to 8-week rearing period. In the majority of flocks, sampled every 2 weeks throughout production, Campylobacter-positive fecal and cecal samples were not detected until 4 to 8 weeks of age. In only six of the flocks were environmental samples found to be positive before shedding of Campylobacter was detected in the birds. Even in some of the Campylobacter-negative flocks, contamination of the rearing environment was positive for Campylobacter but did not result in the birds subsequently excreting the organism. These findings are discussed in relation to U.S. husbandry practices and present uncertainty about sources of Campylobacter infection for poultry flocks. Birds were often transported to the processing plant in coops that were already contaminated with Campylobacter, and the organisms were sometimes found in samples of scald water and chill water. After chilling, the proportions of Campylobacter-positive carcasses from different producers ranged from 21.0 to 40.9%, which is lower than in other studies, and possible reasons are considered.

Age Factors↗
PubMed2001

Occurrence of Clostridium perfringens in the broiler chicken processing plant as determined by recovery in iron milk medium.

Over 30 years ago, Clostridium perfringens was reported as a contaminant of the processing plant and processed carcasses of broiler chickens. Poultry processing procedures and methods for detecting C. perfringens have changed since that time. Therefore, a study was conducted to determine the incidence and numbers of C. perfringens in the water of the scald tank, the water of the chill tank, and the rinse water of the processed carcasses from modern broiler chicken processing plants. In trial 1, collected samples were inoculated into iron milk medium (IMM) and incubated at 46 degrees C for 18 h (the traditional method) or at 37 degrees C for 3 h followed by incubation at 46 degrees C for 15 h (an injury recovery method). Each of three preselected broiler chicken flocks from two integrators were the first processed for that processing shift. The overall incidence of confirmed C. perfringens in samples associated with the three flocks was 40% of postprocessing scald water samples, 13% of preprocessing chill water samples, 13% of postprocessing chill water samples, and 19% of carcass rinses. The incidence of C. perfringens in samples incubated in IMM using the injury recovery procedure was significantly higher than in samples incubated in IMM by the traditional method, but only when all samples associated with the three flocks were pooled. In trial 2, water samples from each tank of a three-tank counterflow scalder, water samples from the prechill and chill tank, and samples of carcass rinses were collected in the middle of a processing shift during multiple visits to a processing plant. Samples were inoculated into IMM with neomycin and polymyxin B sulfate (IMMA) and incubated using the traditional and injury recovery procedures. The incidence of C. perfringens in water samples was 100% from scald tank 1, 100% from scald tank 2, 100% from scald tank 3, 88% from the prechill tank, and 63% from the chill tank. The incidence in carcass rinse samples was 67%. The mean most probably number (MPN) of C. perfringens for contaminated samples decreased from log10 5.07/100 ml of water in scald tank 1 to log10 1.26/100 ml of water in the chill tank. The mean MPN in carcass rinse samples was log10 1.20 C. perfringens per 100 ml. The incidence and mean MPN of C. perfringens in these samples after heat shock at 75 degrees C for 20 min was somewhat less, but high enough to indicate that much of the contamination arises from heat-resistant spores of this organism. In trial 2, there were no differences in incidence and MPN of C. perfringens in samples incubated in IMMA with the traditional method or the injury recovery method.

Animals↗
PubMed2000

Postsynaptic targeting of MAGUKs mediated by distinct N-terminal domains.

Postsynaptic targeting of PSD-95 has been extensively studied; however, little is known about how other MAGUKs are localized. Proper targeting of PSD-95 requires dual palmitoylation of an N-terminal motif. We now find that the N-termini of closely related PSD-93 and SAP-102 are also involved in postsynaptic targeting. PSD-93 is N-terminally palmitoylated; however, unlike PSD-95, palmitoylation does not explain the necessity of the N-terminus for PSD-93 postsynaptic targeting. Furthermore, when the N-terminus of PSD-95 is replaced with the first 30 or 64, but not the first 10, amino acids of PSD-93, the chimera is targeted to postsynaptic sites independent of palmitoylation. Similarly, when the N-terminus of PSD-95 is replaced with the non-palmitoylated N-terminus of SAP-102, postsynaptic targeting is maintained. These results suggest that MAGUKs contain diverse signals within their N-termini for postsynaptic targeting.

Amino Acid Sequence↗
PubMed2000

Ion channel clustering by membrane-associated guanylate kinases. Differential regulation by N-terminal lipid and metal binding motifs.

The postsynaptic density protein PSD-95 and related membrane-associated guanylate kinase (MAGUK) proteins assemble signal transduction complexes at sites of cell-cell contact including synapses. Whereas PSD-95 and PSD-93 occur only at postsynaptic sites in hippocampal neurons, SAP-102 also occurs in axons. In heterologous cells, PSD-95 and PSD-93 mediate cell surface ion channel clustering, but SAP-102 and SAP-97 do not. This selective ion channel clustering activity by MAGUKs is explained by differential palmitoylation, as PSD-93 and PSD-95 are palmitoylated though SAP-97, and SAP-102 are not. Rather than being palmitoylated, we find that N-terminal cysteines from SAP-102 tightly bind to zinc. And, appending the N terminus of SAP-102 to PSD-95 results in localization of the chimera to both axons and dendrites. These data suggest that lipid modifications and heavy metal associations with the N termini of MAGUKs mediate differential functions and subcellular localizations of these synaptic scaffolds.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Synaptic targeting of the postsynaptic density protein PSD-95 mediated by a tyrosine-based trafficking signal.

Synaptic function requires proper localization of proteins at synaptic sites. Targeting of the postsynaptic density protein 95 (PSD-95) relies on multiple signals within the protein, including twelve C-terminal amino acids. We now show that this C-terminal targeting domain of PSD-95 mediates postsynaptic localization through a short tyrosine-based motif followed by a pair of hydrophobic amino acids. Consistent with a role in cellular trafficking, the tyrosine motif resembles the canonical motif for interactions with clathrin adaptor proteins. In fact, we find that the C-terminal targeting domain of PSD-95 is sufficient to mediate clathrin-dependent endocytosis when appended to a transmembrane protein. Furthermore, systematic mutagenesis reveals that endocytosis mediated by this domain depends on both the tyrosine motif and the dihydrophobic amino acid pair. Thus, postsynaptic targeting of PSD-95 requires a tyrosine-based signal that can mediate clathrin-coated vesicle formation.

Amino Acid Sequence↗
PubMed2000

Dual palmitoylation of PSD-95 mediates its vesiculotubular sorting, postsynaptic targeting, and ion channel clustering.

Postsynaptic density-95 (PSD-95/SAP-90) is a palmitoylated peripheral membrane protein that scaffolds ion channels at excitatory synapses. To elucidate mechanisms for postsynaptic ion channel clustering, we analyzed the cellular trafficking of PSD-95. We find that PSD-95 transiently associates with a perinuclear membranous compartment and traffics with vesiculotubular structures, which migrate in a microtubule-dependent manner. Trafficking of PSD-95 with these vesiculotubular structures requires dual palmitoylation, which is specified by five consecutive hydrophobic residues at the NH(2) terminus. Mutations that disrupt dual palmitoylation of PSD-95 block both ion channel clustering by PSD-95 and its synaptic targeting. Replacing the palmitoylated NH(2) terminus of PSD-95 with alternative palmitoylation motifs at either the NH(2) or COOH termini restores ion channel clustering also induces postsynaptic targeting, respectively. In brain, we find that PSD-95 occurs not only at PSDs but also in association with intracellular smooth tubular structures in dendrites and spines. These data imply that PSD-95 is an itinerant vesicular protein; initial targeting of PSD-95 to an intracellular membrane compartment may participate in postsynaptic ion channel clustering by PSD-95.

Animals↗
PubMed2000

Colonization of the intestinal tract by Clostridium perfringens and fecal shedding in diet-stressed and unstressed broiler chickens.

Commercial broiler chicks were given a three-strain composite of bacitracin-resistant Clostridium perfringens by oral gavage and were sampled periodically to determine the dynamics of C. perfringens colonization of the intestinal tract of broiler chickens and fecal shedding. After gavage, the chicks were divided into two groups and placed in isolators, one group received a traditional corn-based diet, and the other group received the same diet supplemented with 50% rye to place the birds under dietary stress. The numbers of bacitracin-resistant C. perfringens in various parts of the intestinal tract, liver, and feces were determined using a selective plating medium containing bacitracin. In chickens on the corn-based or 50% rye diet, C. perfringens was isolated infrequently from the various parts of the intestinal tract, liver, or fecal droppings during the first 36 h following the last gavage. For the 24 chickens on each of the two diets that were sampled from 2 to 21 d after gavage, C. perfringens was recovered more frequently from the crop, proventriculus, duodenum, jejunum, ileum, ceca, and feces, but not the gizzard, of birds on the 50% rye diet as compared to those on the corn-based diet. From 2 to 21 d, the intestinal numbers of C. perfringens in contaminated birds on the corn-based diet were < or =log10 6.2 and did not increase in any portion of the intestinal tract. Numbers of C. perfringens during this time period increased 3.3 to 4.9 log factors in the ileum, ceca, and feces of birds on the 50% rye diet to as high as log10 7.1 to 7.9 at 21 d. This study confirms that addition of rye to the diet of chickens can increase the numbers of C. perfringens in the ceca of broiler chickens and extends these findings to demonstrate increases in the numbers and frequency of recovery of C. perfringens in other parts of the intestinal tract.

Animal Feed↗
PubMed1999

Synaptic targeting of the postsynaptic density protein PSD-95 mediated by lipid and protein motifs.

During synaptic development, proteins aggregate at specialized pre- and postsynaptic structures. Mechanisms that mediate protein clustering at these sites remain unknown. To investigate this process, we analyzed synaptic targeting of a postsynaptic density protein, PSD-95, by expressing green fluorescent protein- (GFP-) tagged PSD-95 in cultured hippocampal neurons. We find that postsynaptic clustering relies on three elements of PSD-95: N-terminal palmitoylation, the first two PDZ domains, and a C-terminal targeting motif. In contrast, disruptions of PDZ3, SH3, or guanylate kinase (GK) domains do not affect synaptic targeting. Palmitoylation is sufficient to target the diffusely expressed SAP-97 to synapses, and palmitoylation cannot be replaced with alternative membrane association motifs, suggesting that a specialized synaptic lipid environment mediates postsynaptic clustering. The requirements for PDZ domains and a C-terminal domain of PSD-95 indicate that protein-protein interactions cooperate with lipid interactions in synaptic targeting.

Animals↗
PubMed1998

Immunocytochemical localization of the postsynaptic density protein PSD-95 in the mammalian retina.

Synapse-associated proteins are the scaffold for the selective aggregation of ion channels at synapses; they provide the link to cytoskeletal elements and possibly are involved with the regulation of synaptic efficacy by electrical activity. The localization of the postsynaptic density protein PSD-95 was studied in different mammalian retinae (rat, monkey, and tree shrew) by using immunocytochemical methods. Immunofluorescence for PSD-95 was most prominent in the outer plexiform layer (OPL). The axon terminals of rods and cones, the rod spherules and cone pedicles, were strongly labeled. Electron microscopy, using preembedding immunocytochemistry, showed PSD-95 localized presynaptically within the photoreceptor terminals. Distinct PSD-95 labeling was also present in the inner plexiform layer (IPL). It had a punctate appearance suggesting the synaptic clustering of PSD-95 in the IPL. Electron microscopy showed that PSD-95 was concentrated in processes that were postsynaptic at bipolar cell ribbon synapses (dyads). As a rule, only one of the two postsynaptic members of the dyad was labeled for PSD-95. Double-labeling experiments were performed for PSD-95 and for SAP 102 or PSD-93, respectively, two other members of the family of synapse-associated proteins. All three were found to be colocalized in the synaptic hot spots in the IPL. In the OPL, however, PSD-95 and PSD-93 were found presynaptically, whereas SAP 102 was located postsynaptically at photoreceptor synapses. Double-labeling experiments also were performed for PSD-95 and for the NR1 subunit of the NMDA receptor. They were found to be colocalized in synaptic hot spots in the IPL.

Animals↗
PubMed1998

In vitro attachment of Salmonella typhimurium to chicken cecal mucus: effect of cations and pretreatment with Lactobacillus spp. isolated from the intestinal tracts of chickens.

The attachment of radiolabeled Salmonella typhimurium 333/O cells to immobilized cecal mucus from specific-pathogen-free leghorn chickens was determined in the presence of D-mannose. The attachment of S. typhimurium was inhibited by the chelating agents EDTA and citrate and by lanthanum but was enhanced in the presence of the calcium, barium, and manganese divalent cations. Summary findings of the effect of lectins are included. Attachment of lactobacilli, previously isolated from the intestines of chickens, to mucus was also enhanced by calcium and inhibited by chelators. The pretreatment of immobilized mucus with portions of cultures of five of eight strains of lactobacilli inhibited subsequent attachment of the S. typhimurium strain. Spent culture supernatant fluid and/or washed cells from these cultures inhibited attachment, and inhibition was enhanced by preheating the cells or supernatant fluid at 80 degrees C. Results indicate that S. typhimurium mucus attachment not involving mannosyl-dependent receptors is influenced by presence of cations. Lactobacillus spp. isolated from the intestinal tracts of chickens produce cellular and cell-free components that inhibit this form of attachment to chicken intestinal mucus.

Animals↗
PubMed1996

Cloning and characterization of postsynaptic density 93, a nitric oxide synthase interacting protein.

Nitric oxide (NO) formation in brain is regulated by the calcium/calmodulin dependence of neuronal NO synthase (nNOS). Calcium influx through NMDA-type glutamate receptors is efficiently coupled to nNOS activity, whereas many other intracellular calcium pathways are poorly coupled. To elucidate possible mechanisms responsible for this coupling, we performed yeast two-hybrid screening to identify proteins that interact with nNOS. Two nNOS interacting proteins were identified: the postsynaptic density proteins PSD-93 and PSD-95. Here, we report the cloning and characterization of PSD-93. PSD-93 is expressed in discrete neuronal populations as well as in specific non-neuronal cells, and it exhibits complex molecular diversity attributable to tissue-specific alternative splicing. PSD-93, like PSD-95, binds to nNOS and to the NMDA receptor 2B. PSD-93, however, is unique among PSD-95/SAP-90 family members in its expression in Purkinje neuron cell bodies and dendrites. We also demonstrate that the PDZ domain at the N terminus of nNOS is required, but it is not sufficient for interaction with PSD-93/95. Given that PSD-93 and PSD-95 each contain multiple potential binding sites for nNOS and the NMDA receptor, complexes involving oligomers of PSD-93/95 may help account for the functional as well as the physical coupling of nNOS to NMDA receptors.

Alternative Splicing↗
PubMed1996

Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains.

Neuronal nitric oxide synthase (nNOS) is concentrated at synaptic junctions in brain and motor endplates in skeletal muscle. Here, we show that the N-terminus of nNOS, which contains a PDZ protein motif, interacts with similar motifs in postsynaptic density-95 protein (PSD-95) and a related novel protein, PSD-93.nNOS and PSD-95 are coexpressed in numerous neuronal populations, and a PSD-95/nNOS complex occurs in cerebellum. PDZ domain interactions also mediate binding of nNOS to skeletal muscle syntrophin, a dystrophin-associated protein. nNOS isoforms lacking a PDZ domain, identified in nNOSdelta/delta mutant mice, do not associate with PSD-95 in brain or with skeletal muscle sarcolemma. Interaction of PDZ-containing domains therefore mediates synaptic association of nNOS and may play a more general role in formation of macromolecular signaling complexes.

Amino Acid Sequence↗