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Biomedical subjects

W L Smith

Publications and source records attributed to W L Smith.

At least 19 recordsLinked to original sources
PubMed2005

Disinfection of dairy and animal farm wastewater with radiofrequency power.

Radiofrequency (RF) power was investigated as a new, physical (nonchemical), thermal process to disinfect wastewater from dairy and animal facilities. Samples (n = 38) from 8 dairy, 2 calf, and 3 swine facilities in California were collected over a 3-yr period and characterized for their dielectric properties, chemical composition, and suitability for thermal processing using RF power. To establish efficacy for disinfection, selected samples were inoculated with high levels (10(6) to 10(9) cfu/mL) of bacterial pathogens such as Salmonella sp., Escherichia coli O157:H7, and Mycobacterium avium ssp. paratuberculosis and processed with an RF prototype system. The capabilities of RF power as a method for thermal disinfection of wastewater were demonstrated when bacteria pathogens were completely and rapidly (<1 min) inactivated when temperatures of 60 to 65 degrees C were achieved. Furthermore, RF technology can be used for large-scale, batch or continuous and portable applications, allowing significant improvements in energy-use efficiencies compared with conventional thermal (surface heating) technologies. Therefore, RF power has potential as an alternative to disinfect dairy/animal farm wastewater before recycling.

Agriculture↗
PubMed2003

The cytokine markers in Staphylococcus aureus mastitis of bovine mammary gland.

TaqMan real time PCR was used to study the transcriptional activity of the bovine IL-2, IL-6, IL-12p40, IFN-gamma, TNF-alpha and granulocyte-monocyte colony stimulating factor of whole milk cells in bovine mammary gland experimentally infected with Staphylococcus aureus. Cytokine transcriptional activity was monitored at 7, 24 and 32 h Post-infection (Pi). IL-12 and TNF-alpha levels were significantly elevated at 24 h Pi followed by sharp decrease at 32 h pi. IL-2 level was decreased at 32 h pi. IL-12 and IFN-gamma showed a significant interaction at 24 h pi. The significant elevations of the IL-12 and TNF-alpha transcriptional level most likely indicate their important role in regulation of the immune responses of bovine mammary gland in S. aureus infection. Depression of IL-2 could reflect the suppressive nature of the S. aureus mastitis.

Animals↗
PubMed2003

The use of spiral plating and microscopic colony counting for the rapid quantitation of Mycobacterium paratuberculosis.

AIMS: To evaluate a spiral plating and microscopic colony counting technique to hasten the quantitation of Mycobacterium paratuberculosis. METHODS AND RESULTS: Broth and milk cultures of M. paratuberculosis were spirally plated onto Middlebrook agar plates and microscopically counted at 8 and 14 days of incubation. The same plates were recounted at 27-28 days of incubation when grossly visible colonies were present. The results were statistically compared with no difference in CFU ml-1 derived from the shorter vs longer incubation times. Other mycobacteria isolates were also plated and microscopically examined and found to be easily distinguishable from M. paratuberculosis. CONCLUSIONS: Microscopic quantitation of spirally plated M. paratuberculosis cultures can be achieved within 8-14 days of plate incubation and compare favourably to counts derived after prolonged incubations. SIGNIFICANCE AND IMPACT OF THE STUDY: The technique could greatly hasten the quantitation of viable M. paratuberculosis.

Colony Count, Microbial↗
PubMed2002

The development and evaluation of a three-dimensional ultrasound-guided breast biopsy apparatus.

We have designed a prototype three-dimensional ultrasound guidance (3D USB) apparatus to improve the breast biopsy procedure. Features from stereotactic mammography and free-hand US-guided biopsy have been combined with 3D US imaging. This breast biopsy apparatus accurately guides a needle into position for the sampling of target tissue. We have evaluated this apparatus in three stages. First, by testing the placement accuracy of a needle in a tissue mimicking phantom. Second, with tissue mimicking phantoms that had embedded lesions for biopsy. Finally, by comparison to free-hand US-guided biopsy, using chicken breast phantoms. The first two stages of evaluation quantified the mechanical biases in the 3D USB apparatus. Compensating for these, a 96% success rate in targeting 3.2 mm "lesions" in chicken breast phantoms was achieved when using the 3D USB apparatus. The expert radiologists performing biopsies with free-hand US guidance achieved a 94.5% success rate. This has proven an equivalence between our apparatus, operated by non-experts, and free-hand biopsy performed by expert radiologists, for 3.2 mm lesions in vitro, with a 95% confidence.

Animals↗
PubMed2001

Different suicide inactivation processes for the peroxidase and cyclooxygenase activities of prostaglandin endoperoxide H synthase-1.

Prostaglandin endoperoxide H synthases (PGHSs)-1 and -2 have a cyclooxygenase (COX) activity involved in forming prostaglandin G2 (PGG2) from arachidonic acid and an associated peroxidase (POX) activity that reduces PGG2 to PGH2. Suicide inactivation processes are observed for both POX and COX reactions. Here we report COX reaction conditions for PGHS-1 under which complete COX inactivation occurs but with > or = 60% retention of POX activity. The rates of POX inactivation were compared for native oPGHS-1 versus Y385F oPGHS-1, a mutant that cannot form the Tyr385 radical of COX Intermediate II; the rates were the same for both native and Y385F oPGHS-1. Our data indicate that a COX Intermediate II/acyl or product complex is the precursor in COX inactivation. However, another species, probably an Intermediate II-like species but with a radical centered on a tyrosine other than Tyr385, is the immediate precursor for POX inactivation.

Animals↗
PubMed2001

Structure of eicosapentaenoic and linoleic acids in the cyclooxygenase site of prostaglandin endoperoxide H synthase-1.

Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) can oxygenate 18-22 carbon polyunsaturated fatty acids, albeit with varying efficiencies. Here we report the crystal structures of eicosapentaenoic acid (EPA, 20:5 n-3) and linoleic acid (LA, 18:2 n-6) bound in the cyclooxygenase active site of Co(3+) protoporphyrin IX-reconstituted ovine PGHS-1 (Co(3+)-oPGHS-1) and compare the effects of active site substitutions on the rates of oxygenation of EPA, LA, and arachidonic acid (AA). Both EPA and LA bind in the active site with orientations similar to those seen previously with AA and dihomo-gamma-linolenic acid (DHLA). For EPA, the presence of an additional double bond (C-17/C-18) causes this substrate to bind in a "strained" conformation in which C-13 is misaligned with respect to Tyr-385, the residue that abstracts hydrogen from substrate fatty acids. Presumably, this misalignment is responsible for the low rate of EPA oxygenation. For LA, the carboxyl half binds in a more extended configuration than AA, which results in positioning C-11 next to Tyr-385. Val-349 and Ser-530, recently identified as important determinants for efficient oxygenation of DHLA by PGHS-1, play similar roles in the oxygenation of EPA and LA. Approximately 750- and 175-fold reductions in the oxygenation efficiency of EPA and LA were observed with V349A oPGHS-1, compared with a 2-fold change for AA. Val-349 contacts C-2 and C-3 of EPA and C-4 of LA orienting the carboxyl halves of these substrates so that the omega-ends are aligned properly for hydrogen abstraction. An S530T substitution decreases the V(max)/K(m) of EPA and LA by 375- and 140-fold. Ser-530 makes six contacts with EPA and four with LA involving C-8 through C-16; these interactions influence the alignment of the substrate for hydrogen abstraction. Interestingly, replacement of Phe-205 increases the volume of the cyclooxygenase site allowing EPA to be oxygenated more efficiently than with native oPGHS-1.

Arachidonic Acid↗
PubMed2001

Three-dimensional ultrasound-guided core needle breast biopsy.

A new core needle breast biopsy system guided by 3-D ultrasound (US) is proposed. Our device provides rapid imaging and real-time guidance, as well as breast stabilization and a needle guidance apparatus using 3-D imaging. The targeting accuracy of our device was tested by inserting a 14-gauge biopsy needle into agar phantoms under 3-D US guidance. A total of 18 0.8-mm stainless-steel beads embedded in the phantoms defined each of the four target positions tested. Positioning accuracy was calculated by comparing needle tip position to the preinsertion bead position, as measured by three observers three times each on 3-D US. The interobserver standard error of measurement was no more than 0.14 mm for the beads and 0.27 mm for the needle tips. A 3-D principal component analysis was performed to obtain the population distribution of needle tip position relative to the target beads for the four target positions. The 3-D 95% confidence intervals were found to have total widths ranging from 0.43 to 1.71 mm, depending on direction and bead position.

Biopsy, Needle↗
PubMed2001

Objective tinnitus in children.

OBJECTIVE: To discuss the various symptoms and causes of objective tinnitus in children. STUDY DESIGN: Retrospective case review. PATIENTS: Five children who had audible signals emanating from their ears caused by audible spontaneous otoacoustic emissions, palatal myoclonus, arteriovenous malformation, and acoustic trauma. MAIN OUTCOME MEASURE: The tinnitus reported by the patients was linked to acoustic signals that could be measured objectively or heard by the examiner. RESULTS: Four of the five children had essentially normal hearing. The one child who demonstrated a hearing loss audiometrically was thought to have normal hearing sensitivity, but his intense roaring objective tinnitus appeared to mask his low-frequency thresholds. All five patients had measurable acoustic signals in the ear canal that matched the patients' descriptions of their tinnitus. CONCLUSION: The cases illustrate how the objective tinnitus was diagnosed and measured, how a treatment algorithm was applied, and the expected results of treatment. In addition, the cases provide support for the use of psychologic counseling throughout the examination and treatment of objective tinnitus in children.

Adolescent↗
PubMed2000

Quantitation of bovine cytokine mRNA in milk cells of healthy cattle by real-time TaqMan polymerase chain reaction.

Here we present a novel methodology to quantitate bovine cytokines and growth factors contributing to immunity against bacterial infections of the mammary gland in cattle. Real-time TaqMan PCR systems were developed to overcome limitations of conventional quantitative PCR methods. The TaqMan method is based on the cleavage of fluorescent dye-labeled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by an automated spectrophotometer integrated in a sequence detection system (Applied Biosystems, Foster City, CA). The bovine-specific TaqMan probes were designed to encompass an intron, thus allowing differentiation between complementary DNA (cDNA) and genomic DNA (gDNA) amplification products. Quantitative analysis of cytokine cDNA was performed in comparison to bovine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Messenger RNA (mRNA) from the universally expressed housekeeping gene GAPDH proved to be useful as an amplification control and allowed for correction of variations in different numbers of cells in the starting material, in the efficiencies of RNA extraction and reverse transcription. With this method, high-throughput analysis of large numbers of samples was possible within a short time. In addition, decreasing the numbers of working steps shortened the time for analysis and increased accuracy. Profiles of cytokines (interleukin (IL)-2, IL-6, IL-8, IL-12 p40, TNF-alpha, IFN-gamma) and granulocyte-macrophage colony stimulating factor (GM-CSF) were established in normal lactating cattle. Differences of cytokine profiles obtained with the real-time TaqMan PCR system and conventional methods are discussed.

Animals↗
PubMed2000

Prostaglandin endoperoxide H synthase-1: the functions of cyclooxygenase active site residues in the binding, positioning, and oxygenation of arachidonic acid.

Prostaglandin endoperoxide H synthases (PGHSs) catalyze the committed step in the biosynthesis of prostaglandins and thromboxane, the conversion of arachidonic acid, two molecules of O(2), and two electrons to prostaglandin endoperoxide H(2) (PGH(2)). Formation of PGH(2) involves an initial oxygenation of arachidonate to yield PGG(2) catalyzed by the cyclooxygenase activity of the enzyme and then a reduction of the 15-hydroperoxyl group of PGG(2) to form PGH(2) catalyzed by the peroxidase activity. The cyclooxygenase active site is a hydrophobic channel that protrudes from the membrane binding domain into the core of the globular domain of PGHS. In the crystal structure of Co(3+)-heme ovine PGHS-1 complexed with arachidonic acid, 19 cyclooxygenase active site residues are predicted to make a total of 50 contacts with the substrate (Malkowski, M. G, Ginell, S., Smith, W. L., and Garavito, R. M. (2000) Science 289, 1933-1937); two of these are hydrophilic, and 48 involve hydrophobic interactions. We performed mutational analyses to determine the roles of 14 of these residues and 4 other closely neighboring residues in arachidonate binding and oxygenation. Mutants were analyzed for peroxidase and cyclooxygenase activity, and the products formed by various mutants were characterized. Overall, the results indicate that cyclooxygenase active site residues of PGHS-1 fall into five functional categories as follows: (a) residues directly involved in hydrogen abstraction from C-13 of arachidonate (Tyr-385); (b) residues essential for positioning C-13 of arachidonate for hydrogen abstraction (Gly-533 and Tyr-348); (c) residues critical for high affinity arachidonate binding (Arg-120); (d) residues critical for positioning arachidonate in a conformation so that when hydrogen abstraction does occur the molecule is optimally arranged to yield PGG(2) versus monohydroperoxy acid products (Val-349, Trp-387, and Leu-534); and (e) all other active site residues, which individually make less but measurable contributions to optimal catalytic efficiency.

Amino Acids↗
PubMed2000

Mutational and X-ray crystallographic analysis of the interaction of dihomo-gamma -linolenic acid with prostaglandin endoperoxide H synthases.

Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) catalyze the committed step in prostaglandin biosynthesis. Both isozymes can oxygenate a variety of related polyunsaturated fatty acids. We report here the x-ray crystal structure of dihomo-gamma-linolenic acid (DHLA) in the cyclooxygenase site of PGHS-1 and the effects of active site substitutions on the oxygenation of DHLA, and we compare these results to those obtained previously with arachidonic acid (AA). DHLA is bound within the cyclooxygenase site in the same overall L-shaped conformation as AA. C-1 and C-11 through C-20 are in the same positions for both substrates, but the positions of C-2 through C-10 differ by up to 1.74 A. In general, substitutions of active site residues caused parallel changes in the oxygenation of both AA and DHLA. Two significant exceptions were Val-349 and Ser-530. A V349A substitution caused an 800-fold decrease in the V(max)/K(m) for DHLA but less than a 2-fold change with AA; kinetic evidence indicates that C-13 of DHLA is improperly positioned with respect to Tyr-385 in the V349A mutant thereby preventing efficient hydrogen abstraction. Val-349 contacts C-5 of DHLA and appears to serve as a structural bumper positioning the carboxyl half of DHLA, which, in turn, positions properly the omega-half of this substrate. A V349A substitution in PGHS-2 has similar, minor effects on the rates of oxygenation of AA and DHLA. Thus, Val-349 is a major determinant of substrate specificity for PGHS-1 but not for PGHS-2. Ser-530 also influences the substrate specificity of PGHS-1; an S530T substitution causes 40- and 750-fold decreases in oxygenation efficiencies for AA and DHLA, respectively.

8,11,14-Eicosatrienoic Acid↗
PubMed2000

The laterophysic connection in chaetodontid butterflyfish: morphological variation and speculations on sensory function.

The laterophysic connection is a novel specialization in chaetodontid butterflyfish, in which paired diverticula of the swim-bladder ('horns') extend anteriorly and approach or directly contact a medial fossa in the lateral line canal contained within the supracleithrum. This study examined the morphology of the laterophysic connection in eight ecologically diverse species belonging to five subgenera within Chaetodon. Two types of laterophysic connections, indirect and direct, were found among Chaetodon species. Intraspecific variation (including sexual dimorphism) in the morphology of the laterophysic connection was not found. The type of laterophysic connection is not correlated with ecological characteristics among Chaetodon species, but appears to be correlated with subgeneric affinities of Chaetodon species. The presence of swim-bladder horns probably increases pressure sensitivity to the inner ear. It is suggested that the presence of a direct laterophysic connection, and possibly an indirect laterophysic connection, imparts pressure sensitivity to the lateral line canal system as well.

Air Sacs↗
PubMed2000

The productive conformation of arachidonic acid bound to prostaglandin synthase.

Prostaglandin H synthase-1 and -2 (PGHS-1 and -2) catalyze the committed step in prostaglandin synthesis and are targets for nonsteroidal anti-inflammatory drugs (NSAIDs) like aspirin. We have determined the structure of PGHS-1 at 3 angstrom resolution with arachidonic acid (AA) bound in a chemically productive conformation. The fatty acid adopts an extended L-shaped conformation that positions the 13proS hydrogen of AA for abstraction by tyrosine-385, the likely radical donor. A space also exists for oxygen addition on the antarafacial surface of the carbon in the 11-position (C-11). While this conformation allows endoperoxide formation between C-11 and C-9, it also implies that a subsequent conformational rearrangement must occur to allow formation of the C-8/C-12 bond and to position C-15 for attack by a second molecule of oxygen.

Arachidonic Acid↗
PubMed2000

Peroxidase activity in prostaglandin endoperoxide H synthase-1 occurs with a neutral histidine proximal heme ligand.

Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) convert arachidonic acid to prostaglandin H(2) (PGH(2)), the committed step in prostaglandin and thromboxane formation. Interaction of peroxides with the heme sites in PGHSs generates a tyrosyl radical that catalyzes subsequent cyclooxygenase chemistry. To study the peroxidase reaction of ovine oPGHS-1, we combined spectroscopic and directed mutagenesis data with X-ray crystallographic refinement of the heme site. Optical and Raman spectroscopy of oxidized oPGHS-1 indicate that its heme iron (Fe(3+)) exists exclusively as a high-spin, six-coordinate species in the holoenzyme and in heme-reconstituted apoenzyme. The sixth ligand is most likely water. The cyanide complex of oxidized oPGHS-1 has a six-coordinate, low-spin ferric iron with a v[Fe-CN] frequency at 445 cm(-)(1); a monotonic sensitivity to cyanide isotopomers that indicates the Fe-CN adduct has a linear geometry. The ferrous iron in reduced oPGHS-1 adopts a high-spin, five-coordinate state that is converted to a six-coordinate, low-spin geometry by CO. The low-frequency Raman spectrum of reduced oPGHS-1 reveals two v[Fe-His] frequencies at 206 and 222 cm(-)(1). These vibrations, which disappear upon addition of CO, are consistent with a neutral histidine (His388) as the proximal heme ligand. The refined crystal structure shows that there is a water molecule located between His388 and Tyr504 that can hydrogen bond to both residues. However, substitution of Tyr504 with alanine yields a mutant having 46% of the peroxidase activity of native oPGHS-1, establishing that bonding of Tyr504 to this water is not critical for catalysis. Collectively, our results show that the proximal histidine ligand in oPGHS-1 is electrostatically neutral. Thus, in contrast to most other peroxidases, a strongly basic proximal ligand is not necessary for peroxidase catalysis by oPGHS-1.

Animals↗