Analysis of the lytic activity of the Serpulina hyodysenteriae hemolysin

The hemolysins of Serpulina hyodysenteriae are active at 27 to 40 degrees C and pH 3 to 9 and are unaffected by enzymatic inhibitors. Pore formation was demonstrated by the inhibition of hemolysis with molecules of 2.0 to 2.3 nm in diameter and the release of 86rubidium from erythrocytes without hemoglobin release after exposure to native hemolysin.     

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.     

Specific detection of the genus Serpulina, S. hyodysenteriae and S. pilosicoliin porcine intestines by fluorescent rRNA in situ hybridization

PURPOSE: Our purpose was to evaluate the utility of spectral imaging for multicolor, multichromosome enumeration in human interphase cell nuclei. METHODS: Chromosome-specific probes labeled with different fluorochromes or nonfluorescent haptens were obtained commercially or prepared in-house. Metaphase spreads, interphase lymphocytes, or blastomeres cells were hybridized with either 7 or 11 distinctly different probes. Following 46 hr of hybridization, slides were washed and detected using either a filter-based quantitative image processing system (QUIPS) developed in-house or a commercial spectral imaging system. RESULTS: The filter-based fluorescence microscope system is preferred for simultaneous detection of up to seven chromosome targets because of its high sensitivity and speed. However, this approach may not be applicable to interphase cells when 11 or more targets need to be discriminated. Interferometer-based spectral imaging with a spectral resolution of approximately 10 nm allows labeling of chromosome-specific DNA probes with fluorochromes having greatly overlapping emission spectra. This leads to increases in the number of fluorochromes or fluorochrome combinations available to score unambiguously chromosomes in interphase nuclei. CONCLUSIONS: Spectral imaging provides a significant improvement over conventional filter-based microscope systems for enumeration of multiple chromosomes in interphase nuclei, although further technical development is necessary in its application to embryonic blastomeres. When applied to preconception/preimplantation genetic diagnosis, presently available probes for spectral imaging are expected to detect abnormalities responsible for 70-80% of spontaneous abortions caused by chromosomal trisomies.     

Identification and characterization of genus-specific epitopes of Serpulina species using monoclonal antibodies

Four murine monoclonal antibodies (mAbs) designated as C9E8, A10, G12, and G8 which recognized both Serpulina hyodysenteriae and S. innocens were produced and characterized. The mAbs reacted with whole cell antigens in ELISA, indirect immunofluorescence and immunoblot assays. The mAbs did not show any cross reactivity in rapid dot ELISA or immunoblot assay with Leptospira icterohemorrhagiae, Campylobacter jejuni and Escherichia coli. Treatment of whole cell suspension with proteinase K and sodium periodate indicated that the reacting epitopes of the mAbs were protein in nature. The genus-specific antigens were identified as heat-stable proteins with molecular weight in the range of 26 to 45 kDa. Immunofluorescence and immunogold labelling studies showed that the antibody-binding epitopes were exposed on the outer-surface of the spirochaetal cell wall. The mAbs inhibited growth of reference strains of both S. hyodysenteriae and S. innocens in vitro but failed to cause agglutination. The detection of spirochaetal forms directly in fecal smears or paraffin-embeded tissue sections from experimentally infected pigs indicated that such mAbs were potentially useful for the diagnosis of swine spirochaetosis. This is the first report of mAbs identifying and characterizing common antigens of porcine Serpulina.     

Fatty acids stimulate trehalose synthesis in trophocytes of the cockroach (Periplaneta americana) fat body

Trophocytes from the disaggregated fat body of the cockroach (Periplaneta americana) respond to synthetic hypertrehalosemic hormone (HTH) by increasing the rate of trehalose synthesis. The cells give a similar response when incubated with stearic, oleic, linoleic, or arachidonic acid. A maximal increase in trehalose synthesis was obtained with 1-10 microM fatty acids. Synthesis of trehalose by the trophocytes was also increased by 1 microM prostaglandin F2alpha to nearly the same extent as that evoked by HTH. Furthermore, the data show that the trophocytes are capable of converting linoleic acid into arachidonic acid. This suggests that the cells may convert arachidonic acid, formed from the linoleic acid released by the action of HTH, to a prostaglandin which serves as an integral part of the hypertrehalosemic mechanism.     

Learning in the intact cockroach (Periplaneta americana) when placed in a punishment situation.

Examined whether 40 intact cockroaches, Periplaneta americana, in a yoked-control punishment paradigm exhibit escape or avoidance learning, or both. Evidence for avoidance learning was unequivocal. Escape learning was not found. Asymptotic avoidance learning was found by 1.5 min. of training. The advantages of defining learning by experimental-control differences developing during training are discussed. (15 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Fine structure of the first optic ganglion (lamina) of the cockroach, Periplaneta americana

The stuctural organization of the first optic ganglion (lamina) of the cockroach (Periplaneta americana) was investigated by the use of light and electron microscopy. Each compound eye of the cockroach is composed of up to 2000 visual units (ommatidia) of the fused rhabdom type. The ommatidia themselves consist of eight receptor cells which terminate as axons in either the first or second optic ganglion. Three different short visual fibre types end in two separate strata in the lamina, and one long fibre type ends in the second optic ganglion. Monopolar second-order neurons with wide field branching patterns in the middle stratum of the first synaptic region have postsynaptic contacts with sort visual fibres. Horizontal fibre elements with branching patterns at different levels of the lamina apparently from three horizontal plexuses with presynaptic and/or postsynaptic connections to first-and second-order neurons. The lack of well-organized fibre cartridges containing a constant number of first and second order neurons in each fascicle and the presence of only unistratified wide field monopolar cells could represent, as compared to other insect orders, a primitive stage in the development of the first optic ganglion.     

Disposal of aerobic treated swine waste by refeeding to finishing swine (author's transl)

Simple aerobic treatment of swine waste did not increase the production of microorganismprotein (pure protein). Addition of available energy resulted in a small increase of pure protein: During continuous aerobic treatment 20 g pure protein were produced by 100 g starch, during discontinuous aerobic treatment 10 g pure protein by 100 g organic matter from molasses (mean values). Comparative feeding trials with 90% resp. 80% feed restricted finishing swine without and with added swine waste (continuous treated without supplements resp. discontinuous treated with addition of yeasts and molasses) had as results no improvement in body-weight-gain but a 12% resp. 17% deterioration in feed conversion with 13% less bodyfat formation. Weight gains of both groups with restricted feed (without and with added swine waste) was 22% less in comparison to full fed swine. Losses at slaughter of swine in the group with feed restriction and added swine waste were partly due to higher intestine contents higher than that of swine with feed restriction and without added swine waste. Higher weights of livers and kidneys in swine of the "waste group" counts for a possibly higher metabolism-stress.     

Experiences with segregated early weaning (SEW) in of swine

The ongoing trend towards SEW has arrived the eastern European countries as well. In order to examine the effect of 17 days weaning on production in a large pig production unit there were the following evaluations, carried out in the following steps: Step one: Two groups of sows were randomly formed before weaning: Group one (90 sows) were weaned at day 21 of lactation. Group two (109 sows) were weaned at day 17 of lactation. The following parameters were evaluated: A: Weaning to estrus interval in days B: Liveborn litter size at the following parturition Step 2: The number of weaned piglets that were the subject to the next evaluation were not the complete number of the weaned ones from the sows of group one and two, as due to technical reasons some of the animals could not be included in the second step of the evaluation. Group one: 901 piglets conventionally weaned at day 21 of lactation. Group two: 798 piglets SEW weaned at day 17 of lactation. The following parameters were evaluated during 6 weeks after weaning: C: Average daily weight (ADG) gain, measured weekly in gram in each group D: Mortality in percent in each group In step 1 the following results were evaluated: the sows in group one showed a significant shorter (p < 0.05) weaning to estrus interval when compared to the sows in group two (5.65 +/- 2.30 vs. 6.38 +/- 2.31), but there was no significant difference regarding liveborn litter size between the groups (10.87 +/- 2.01 vs. 10.74 +/- 2.22). Regarding step 2 the group two (SEW) showed, when compared to the group one (21 day weaning) during the first three weeks after weaning lesser weekly measured daily gains (185 +/- 13; 184 +/- 41; 361 +/- 23 vs. 201 +/- 11; 202 +/- 21; 365 +/- 32). Following the third week after weaning there existed a significant (p < 0.05) better weekly measured average daily gain in the SEW weaned group (group 2) when compared to group one (401 +/- 34; 412 +/- 30; 439 +/- 41 vs. 433 +/- 29; 465 +/- 31; 543 +/- 29). Regarding mortality both of the groups revealed a very low percent of losses (group one 0.89% vs. group two 0.75%), which differences were not significant. Step 3: retrospective evaluation of culling of the sows. Group one (412 sows) were SEW weaned in three consecutive weaning at day 14-17 of lactation. Group two (597 sows) were weaned in three consecutive weaning at day 21-28 of lactation. The reasons for culling included no observed postweaning estrus, repeat breeders, abort, small litter size, locomotor problems, periparturient diseases (PDC/MMA), small weaning litter weight, old age, urogenital diseases, overweight. The calculated average "Culling Rate" showed no significant differences between the groups (46.4% in group one vs. 47.1% in the group two).     

Complete amino acid sequence of chitinase-A from leaves of pokeweed (Phytolacca americana)

The complete amino acid sequence of pokeweed leaf chitinase-A was determined. First all 11 tryptic peptides from the reduced and S-carboxymethylated form of the enzyme were sequenced. Then the same form of the enzyme was cleaved with cyanogen bromide, giving three fragments. The fragments were digested with chymotrypsin or Staphylococcus aureus V8 protease. Last, the 11 tryptic peptides were put in order. Of seven cysteine residues, six were linked by disulfide bonds (between Cys25 and Cys74, Cys89 and Cys98, and Cys195 and Cys208); Cys176 was free. The enzyme consisted of 208 amino acid residues and had a molecular weight of 22,391. It consisted of only one polypeptide chain without a chitin-binding domain. The length of the chain was almost the same as that of the catalytic domains of class IL chitinases. These findings suggested that this enzyme is a new kind of class IIL chitinase, although its sequence resembles that of catalytic domains of class IL chitinases more than that of the class IIL chitinases reported so far. Discussion on the involvement of specific tryptophan residue in the active site of PLC-A is also given based on the sequence similarity with rye seed chitinase-c.     

Quinoxaline-2-carboxylic acid (QCA) in swine liver and muscle

The concentration of quinoxaline-2-carboxylic acid (QCA) determined by HPLC after alkaline hydrolysis of liver and muscle of swine, ranged from < 3 ng/g to 45.3 ng/g in liver, and from < 3 ng/g to 10.8 ng/g in muscle samples. After the 77th day of therapy QCA was found in samples of liver (9.7 ng/g). Recoveries obtained for both liver and muscle were 70% at 5 ng/g, 77% and 75% respectively at 10 ng/g, and 90% for both liver and muscle at 30 ng/g. This experiment was performed within the frame of the National Monitoring Programme of Residues in Animal Tissues in the Republic of Croatia.     

Ketorolac (Toradol) as an analgesic in swine following transluminal coronary angioplasty

The arch forms of 38 cases (53 nonextraction and 23 extraction arches) in which expansion, while maintaining arch form, was the objective of the practitioner, were analyzed before treatment, after treatment, and an average of 6 to 8 years after retention. The cubic spline was used to fit a curve representing arch form. By superimposing the spline curves, changes in arch form were analyzed with the variables rebound change (RC), rebound index (RI), rebound number (RN), and stability number (SN). Traditional linear intraarch dimensions were also analyzed. Analysis of variance was used to determine differences between the maxillary and mandibular arches and between the extraction and nonextraction cases. Pearson correlation coefficients between spline variables and arch width variables were also computed. There was significantly more expansion in the maxillary arch than the mandibular arch during treatment, irrespective of extraction or nonextraction strategies. In the nonextraction cases, a greater amount of net expansion was achieved for all dimensions for the maxillary arch as compared with the mandibular arch. Overall, a relatively high stability in arch form was found. The findings suggest that stability may not be related to the amount of change produced during treatment. Significant expansion can be gained throughout the premolar regions and may be expected to be stable. The order of greatest net arch width gained was for the second premolars followed by first premolars, molars, and then the canines. The intercanine widths for both arches decreased toward pretreatment values, but were more stable in the maxillary arch in nonextraction cases. The cubic spline permits measurement of change in arch form both during treatment and retention periods.     

Overview of the Second International Workshop to define swine cluster of differentiation (CD) antigens

The aim of the Second International Swine Cluster of Differentiation (CD) Workshop, supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters. At the summary meeting of the workshop in July, 1995, revisions in the existing nomenclature for Swine CD were approved, so that the rules are now in accord with those for human and ruminant CD. Swine CD numbers will now be given to clusters of mAb to swine orthologues of human CD molecules when homology is proven by (1) suitable tissue distribution and lymphoid cell subset expression, (2) appropriate molecular mass of the antigen recognized by the mAbs, and (3) reactivity of mAbs with the cloned swine gene products, or cross-reactivity of the mAb on the human gene products. In some cases, this reactivity would not be fully proven, mainly due to the lack of cloned gene products; for these CD antigens, the respective clusters will be assigned by the prefix 'w' which will lead to 'wCD' antigens. As a result of the Second International Swine CD Workshop the assignment of 16 mAb to existing CD groups (CD2a, CD4a, CD5a, wCD6, wCD8, CD14, CD18a, wCD21, wCD25) was confirmed, and 2 mAb to existing swine workshop clusters (SWC). More importantly, for the work on the porcine immune system, was the definition of 5 new swine CD antigens, namely CD3 (recognized by 6 new mAb and 3 epitopes), CD16 (1 new mAb), wCD29 (2 mAb), CD45RA (3 mAb) and CD45RC (1 new mAb). Finally, the demarcation of two new SWC molecules in swine, SWC8 (2 mAb) and SWC9 (2 mAb) was confirmed.     

Quantitative structure-activity studies of octopaminergic 2-(arylimino)thiazolidines and oxazolidines against the nervous system of Periplaneta americana L

The quantitative structure-activity relationship (QSAR) of octopaminergic 2-(arylimino)thiazolidines (AITs) and 2-(arylimino)oxazolidines (AIOs) against the thoracic nerve cord of the American cockroach, Periplaneta americana L., was analysed using reported physicochemical parameters and regression analysis. The more electron-donating, the less bulky at m-position, and the more hydrophobic the substituent, the greater the activity. The plots of observed log Vmax values against calculated log Vmax values having substituents on the m-position deviated downwards from those of compounds having substituents at the 0- and/or p-positions. The more hydrophobic and the more electron-withdrawing the substituent, the greater the activity. AIO with a 2, 3, 4-trichlorophenyl group (58) was more active than its thiazolidine derivative, 2-(2,3,4-trichlorophenylimino)thiazolidine (38) in terms of Vmax:Vmax of 58 was 30% relative to octopamine (OA), whereas that of 38 has been 9% relative to OA, respectively. Superimposition of energy-minimized OA and 58 revealed structural and conformational similarities that might account for the high activity of 58.     

Identification of holocarboxylase synthetase (HCS) proteins in human placenta

The goal of this study was to assess the occurrence of anniversary reactions in Gulf War veterans 2 years after the conclusion of Operation Desert Storm. Subjects were administered questionnaires and asked to identify specific months of best and worst functioning, and months of least or most symptoms of posttraumatic stress disorder (PTSD). Negatively experienced months were compared to documented dates of exposure to traumatic events during the war. Anniversary reactions occurred with a frequency greater than chance and were seen most in individuals exposed to a greater number, and to more severe types, of traumatic events. This suggests that anniversary reactions are etiologically linked to traumatic events and may be a part of the syndrome of PTSD.     

Identification of DNA gyrase inhibitor (GyrI) in Escherichia coli

DNA gyrase is an essential enzyme in DNA replication in Escherichia coli. It mediates the introduction of negative supercoils near oriC, removal of positive supercoils ahead of the growing DNA fork, and separation of the two daughter duplexes. In the course of purifying DNA gyrase from E. coli KL16, we found an 18-kDa protein that inhibited the supercoiling activity of DNA gyrase, and we coined it DNA gyrase inhibitory protein (GyrI). Its NH2-terminal amino acid sequence of 16 residues was determined to be identical to that of a putative gene product (a polypeptide of 157 amino acids) encoded by yeeB (EMBL accession no. U00009) and sbmC (Baquero, M. R., Bouzon, M., Varea, J., and Moreno, F. (1995) Mol. Microbiol. 18, 301-311) of E. coli. Assuming the identity of the gene (gyrI) encoding GyrI with the previously reported genes yeeB and sbmC, we cloned the gene after amplification by polymerase chain reaction and purified the 18-kDa protein from an E. coli strain overexpressing it. The purified 18-kDa protein was confirmed to inhibit the supercoiling activity of DNA gyrase in vitro. In vivo, both overexpression and antisense expression of the gyrI gene induced filamentous growth of cells and suppressed cell proliferation. GyrI protein is the first identified chromosomally nucleoid-encoded regulatory factor of DNA gyrase in E. coli.