Dual flaA1 flaB1 mutant of Serpulina hyodysenteriae expressing periplasmic flagella is severely attenuated in a murine model of swine dysentery

The motility imparted by the periplasmic flagella (PF) of Serpulina hyodysenteriae is thought to play a pivotal role in the enteropathogenicity of this spirochete. The complex PF are composed of multiple class A and class B polypeptides. Isogenic strains containing specifically disrupted flaAl or flaB1 alleles remain capable of expressing PF, although such mutants display aberrant motility in vitro. To further examine the role that these proteins play in the maintenance of periplasmic flagellar structural integrity, motility, and fitness for intestinal colonization, we constructed a novel strain of S. hyodysenteriae which is deficient in both FlaA1 and FlaB1. To facilitate construction of this strain, a chloramphenicol gene cassette, with general application as a selectable marker in prokaryotes, was developed. The cloned flaAl and flaB1 genes were disrupted by replacement of internal fragments with chloramphenicol and kanamycin gene cassettes, respectively. The inactivated flagellar genes were introduced into S. hyodysenteriae, and allelic exchange at the targeted chromosomal flaA1 and flaB1 loci was verified by PCR analysis. Immunoblots or cell lysates with antiserum raised against purified FlaA or FlaB confirmed the absence of the corresponding sheath and core proteins in this dual flagellar mutant. These mutations selectively abolished the expression of the targeted genes without affecting the synthesis of other immunologically related FlaB proteins. The resulting flaA1 flaB1 mutant exhibited altered motility in vitro. Surprisingly, it was capable of assembling periplasmic flagella that were morphologically normal as evidenced by electron microscopy. The virulence of this strain was assessed in a murine model of swine dysentery by determining the incidence of cecal lesions and the persistence of S. hyodysenteriae in the gut. Mice challenged with the wild-type strain or a passage control strain showed a dose-related response to the challenge organism. The dual flagellar mutant was severely attenuated in murine challenge experiments, suggesting that the FlaA1 and FlaB1 proteins are dispensable for flagellar assembly but critical for normal flagellar function and colonization of mucosal surfaces of the gastrointestinal tract. This strain represents the first spirochete engineered to contain specifically defined mutations in more than one genetic locus.     

Gender identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A rapid, simple, and reliable gender determination of human DNA samples was successfully obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Detection sensitivity reached 0.01 ng or less for DNA samples.     

Interkey timing in piano performance and typing

In typing, when the fingers executing two successive movements are on the same hand, the time between keystrokes is longer than when the fingers are on different hands. Biomechanical limitations of the hands are thought to account for this difference. The generality of this finding was explored by investigating skilled pianists' performance of two successive notes. Experiment 1 failed to find comparable differences in timing as a function of the hands involved. Experiment 2, employing both a piano production and a typing task, replicated the previous piano performance results, and revealed that the timing differences in typing were limited to letter sequences requiring fore-aft and lateral finger movements. Experiment 3 extended this finding to piano performance. Together, these findings clarify the nature of biomechanical constraints on skilled manual performance.     

Use of computed radiography in the study of an historic painting

The authors demonstrate the use of radiography in the investigation of an historic painting and describe the potential benefits of computed radiography compared with conventional screen-film radiography. The subject for the comparison was a 16 x 19-foot oil-on-canvas painting, Scipio Africanus Freeing Massiva, by Giovanni Battista Tiepolo. Radiographs of the painting were obtained by using a portable, industrial radiographic unit and both conventional screen-film and photostimulable phosphor plate cassettes. For this investigation, computed radiography had a number of advantages over screen-film radiography, largely due to its wider dynamic range and its capabilities for enhancing the digital images with image processing tools such as magnification, edge enhancement, colorization, and airbrushing. The ability to electronically combine images from the large painting into a single composite image file was extremely valuable, as this technique was much less cumbersome and resulted in much higher quality composite images than could be achieved with conventional radiography. An additional advantage of computed radiography includes the capability to easily archive and transmit these images in a digital format for subsequent review.     

Human cytochrome P4502B6: interindividual hepatic expression, substrate specificity, and role in procarcinogen activation

In situ hybridization was combined with serotonin (5-hydroxytryptamine, 5-HT) or tyrosine hydroxylase immunocytochemistry and with Fluoro-Gold retrograde labeling of bulbo-spinal pathways in order to investigate the expression of GAP-43 mRNA in monoamine cell groups of the adult rat brain stem. Consistent with previous reports, GAP-43 mRNA was observed in serotonin and dopamine cell groups in the pons. In addition, GAP-43 expressing cells were observed in all the major monoamine cell groups in the medulla. Thus the B1, B2 and B3 serotonin cell groups all showed high GAP-43 expression in all contained many GAP-43 expressing serotonin cells with spinal cord projections. The A1, A2, A5 and A6 noradrenaline cell groups also showed high GAP-43 expression, although cells with spinal cord projections were largely restricted to the A5 group and A6 subcoeruleus region. In all areas, GAP-43 expressing cells with spinal cord projections were also observed which were not serotonergic or noradrenergic.     

Effects of pulsed ultrasound on the frog heart: III. The radiation force mechanism

Earlier studies have shown that a single, millisecond duration pulse of ultrasound delivered to the frog heart in vivo during systole can produce a reduction in the developed aortic pressure, while a pulse delivered during diastole can produce a premature ventricular contraction. The threshold for these effects is 5-10 MPa with a 5-ms pulse. Since cardiac tissues respond to mechanical stimulation, the objective of this study was to investigate acoustic radiation force as a possible mechanism for the observed effects of ultrasound on the frog heart. In two experiments, the radiation force exerted on the heart was varied by varying the ultrasonic frequency and the acoustic beam width. Results of these studies indicated that the rate of occurrence of the reduced aortic pressure effect was directly correlated with the magnitude of the radiation force exerted on the heart. A third experiment tested the radiation force mechanism directly by placing an acoustic reflector on the frog heart. The acoustic reflector maximized the radiation force delivered to the heart, but eliminated direct interaction of the ultrasound with the heart and experimentally eliminated heating and cavitation as mechanisms of action. The reduced aortic pressure effect was observed with the reflector on the heart, indicating that radiation force is capable of producing this effect. No premature ventricular contractions were observed with the acoustic reflector over the heart, suggesting that another property of the exposure may be responsible for this bioeffect.     

The 2-His-1-carboxylate facial triad--an emerging structural motif in mononuclear non-heme iron(II) enzymes

PURPOSE: To determine in-line pressures generated in small-bore central venous catheters during power injection of computed tomographic (CT) contrast media. MATERIALS AND METHODS: Five 3.0-7.0-F central venous catheters for pediatric patients were tested at full and half lengths in vitro. In-line pressures were measured during power injection of three contrast media. Rates of injection were increased in steps from 0.1 to 5.0 mL/sec or until a peak pressure of 100 psi (700 kPa) was achieved. The maximum tolerated flow rate was determined with reference to the manufacturer's suggested operating pressure limit for each catheter. RESULTS: At full length, the maximum tolerated flow rates were as follows: 2-3 mL/sec for the large lumen and 1-1.4 mL/sec for the small lumen of the 7.0-F double-lumen catheter; 0.2-0.4 and 0.8-1.2 mL/sec for the 3.0- and 4.0-F peripherally inserted central catheters, respectively; 0.7-1.2 mL/sec for the 6.6-F catheter; and only 0.2 mL/sec for the 4.2-F catheter, which ruptured during testing at higher flow rates. CONCLUSION: Flow rates were documented at which certain small-bore central venous catheters should tolerate power injection of CT contrast media with peak pressures remaining below the manufacturer's recommended operating pressure limits. These data may serve as a guide for clinical use.