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Summary Background Variation in equine caudal cervical spine morphology at C6 and C7 has high prevalence in Warmblood horses and is suspected to be associated with pain in a large mixed‐breed group of horses. At present no data exist on the relationship between radiographic phenotype and clinical presentation in Warmblood horses in a case‐control study. Objectives To establish the frequency of radiographically visible morphologic variation in a large group of Warmblood horses with clinical signs and compare this with a group without clinical signs. We hypothesised that occurrence of morphologic variation in the case group would not differ from the control group, indicating there is no association between clinical signs and morphologic variation. Study design Retrospective case‐control. Methods Radiographic presence or absence of morphologic variation of cervical vertebrae C6 and C7 was recorded in case (n = 245) and control horses (n = 132). Case and control groups were compared by univariable Pearson’s Chi‐square and multivariable logistic regression for measurement variables age, sex, breed, degenerative joint disease and morphologic variation at C6 and C7. Odds ratio and confidence intervals were obtained. A P≤0.05 was considered statistically significant. Results Morphologic variation at C6 and C7 (n = 108/377 = 28.6%; Cases 58/245 = 23.7%; Control 50/132 = 38%) was less frequent in horses with clinical signs in univariable testing (OR 0.48, 95% CI 0.3–0.8, P = 0.001). Age, sex, breed and degenerative joint disease were not retained in the final multivariable logistic regression step whereas morphologic variation remained significantly less present in horses with clinical signs. Main limitations Possible demographic differences between equine clinics. Conclusions Morphologic variation in the caudal cervical spine was detected more frequently in horses without clinical signs. Therefore, radiographic presence of such variation does not necessarily implicate the presence of clinical signs.

Distracted eating can lead to increased food intake, but it is unclear how. We hypothesized that distraction affects the change in motivated responses for food reward after satiation. To investigate this, 38 healthy normal-weight participants (28F, 10M) performed a detection task varying in attentional load (high or low distraction) during fMRI. Simultaneously, they exerted effort for food rewards (sweet or savory) by repeated button presses. Two fMRI runs were separated by outcome devaluation (satiation) of one of the reward outcomes, to assess outcome-sensitive, i.e. goal-directed, responses. Behavioral results showed no effect of distraction on effort for food reward following outcome devaluation. At an uncorrected threshold (pr = - 0.40, p = 0.014) with the effect of distraction on the number of button presses. Specifically, decreased rIFG responses due to distraction related to increased button presses for food reward after satiation, in line with the rIFG’s established role in response inhibition. Furthermore, distraction decreased functional connectivity between the rIFG (seed) and left putamen for valued versus devalued food rewards (pFWE(cluster)https://osf.io/ad2qk.

SummaryDistracted eating is associated with increased food intake and overweight. However, the underlying neurocognitive mechanisms are unknown. To elucidate these mechanisms, 41 healthy normal-weight participants received sips of high- and low-sweet isocaloric chocolate milk, while performing a high- or low-distracting detection task during fMRI on two test days. Subsequently, we measured ad libitum food intake. As expected, a region in the primary taste cortex – located in the insula – responded more to the sweeter drink. Distraction did not affect this right insula sweetness response across the group, but did weaken sweetness-related connectivity of this region to a secondary taste region in the right orbitofrontal cortex. Moreover, distraction-related attenuation of taste processing in the insula predicted increased subsequent ad libitum food intake after distraction between subjects. These results reveal a previously unknown mechanism explaining how distraction during consumption attenuates neural taste processing and increases food intake. The study was preregistered at https://osf.io/vxdhg/register/5771ca429ad5a1020de2872e?view_only=e3207cd6567f41f0a1505e343a64b5aa.

Toll-like receptor-4 (TLR4) pathways are major contributors to pathological inflammatory responses induced by tissue damage. NI-0101 is the first monoclonal antibody (mAb) blocking TLR4 signaling. This activity is independent of the ligand type and concentration, therefore, potentially blocking any TLR4 ligands. A phase I single ascending dose study was conducted in 73 healthy volunteers to evaluate NI-0101 tolerability, preliminary safety, pharmacokinetics (PKs), and pharmacodynamics (PDs), in absence and in presence of a systemic challenge with lipopolysaccharide (LPS), a TLR4 ligand. NI-0101 was well tolerated without safety concern. The PK profile was characterized by a half-life of ∼10 days at high concentrations and by a rapid elimination at low concentrations due to expected target-mediated drug disposition. NI-0101 prevented cytokine release following ex vivo and in vivo LPS administration and prevented the C-reactive protein (CRP) increase and the occurrence of flu-like symptoms expected following the in vivo administration of LPS.

“Eating profiles” can be defined as types of clients distinguished by combinations of food preferences, consumption patterns, and preferences for ambiance. The purpose of this article is to describe the development and initial testing of an instrument to establish eating profiles of residents of nursing homes or elderly homes. We constructed a 35-item, 4-subscale questionnaire. This self-administered, usable instrument derived five eating profiles from clients' perspectives. Insight in eating profiles is important for facility managers to ascertain that the food, dinner service, and ambiance are adequately tailored to the residents' preferences.

An experimental oil adjuvant vaccine was developed against haemorrhagic septicaemia, a disease of cattle and buffalo caused by Pasteurella multocida serotype B and E. Mineral oil, Mercol 52, was used as adjuvant together with Span 85 and Tween 85 as emulsifiers. The vaccine was evaluated by single dose intramuscular immunisation of 1-2 year old buffalo calves. IgG and IgM class antibodies were determined by ELISA. The group of animals immunised with the experimental oil adjuvant vaccine showed a high titre of the IgG class of antibodies measured at 300 days post vaccination. To compare the protective efficacy of the vaccine with the commonly used broth bacterin, another group of buffalo calves was immunised by broth bacterin. This group showed a low level of IgG antibodies. Protection was assessed by challenge with 10(9) viable bacteria of P. multocida type B:2,5 administered subcutaneously, 250 days post vaccination. Animals vaccinated with the experimental oil adjuvant vaccine were fully protected. The other groups of animals, vaccinated with broth bacterin or used as control (non-vaccinated), developed symptoms of haemorrhagic septicaemia. A strong relationship between IgG but not IgM class antibody level and resistance to challenge was observed. The experiment demonstrated that the experimental oil adjuvant vaccine was superior to broth bacterin in providing protection against experimental haemorrhagic septicaemia in young buffalo calves beyond 250 days.

K88ab fimbriae are long polymeric protein structures mainly composed of FaeG proteins. The Escherichia coli K88 periplasmic chaperone FaeE is a homodimer and forms a heterotrimeric complex with the K88 major fimbrial component FaeG in the periplasm. In this study the direct interaction of FaeE and the minor K88 fimbrial subunits FaeH and Fael were investigated. The faeH gene and the fael gene were subcloned in a plNIIIA1-derivative vector containing the faeE gene. SDS-PAGE using normal and gradient gels and immunoblotting revealed that the subcloned genes were expressed in the periplasm. Analyses of periplasmic fractions by native gel electrophoresis and isoelectric focusing (IEF) showed that FaeE and FaeH, as well as FaeE and Fael formed protein complexes. These complexes were isolated and purified by FPLC or IEF and native gel electrophoresis. The stoichiometry of the proteins in these complexes was studied by automated Edman degradation and gel image analysis. The results showed that FaeE and FaeH, and FaeE and Fael formed heterotrimeric E 2 H and E 2 I complexes, respectively. In addition to the E 2 H complex, cells expressing FaeE and FaeH accumulated unbound FaeH in their periplasm. In contrast to the E 2 G complex, the purified E 2 H complex was not stable and was partly dissociated in the experimental conditions used, suggesting that the interaction between FaeE and FaeH is not as strong as the interaction of FaeE and FaeG.

Tn 10 transposon mutagenesis of Escherichia coli producing K99 fimbriae was carried out to identify host factors involved in regulation or biosynthesis of fimbriae. Two chromosomal mutants were obtained that showed a strongly reduced cell surface expression of K99 fimbriae upon colony blotting and ELISA. Analysis by inversed PCR and nucleotide sequencing showed that one mutant (EP14) contained the Tn 10 transposon in rfaQ, affecting theexpression of the rfaQGP gene cluster, whereas the other mutant (EP35) was affected in a, to date, unknown region of the genome. Immunoblotting analysis confirmed a Rd1 type of LPS of mutant strain EP14. These findings for the first time indicated an effect of LPS core biosynthesis on the biogenesis of fimbriae at the cell surface. Preliminary experiments indicated that K99 major subunits, in contrast to K88 subunits, strongly bind LPS molecules.

Glycolipids were prepared from epithelial cells of the small intestine of a newborn calf and assayed for Escherichia coli K99 binding activity on thin-layer chromatograms and in microtiter wells. The bacteria did not bind to any of the non-acid glycolipids, while in the acid fraction several binding-positive glycolipids were detected. The acid glycolipids were isolated and characterized by mass spectrometry, proton NMR spectroscopy and other methods. The following gangliosides were identified, mainly from the epithelial cells from the upper part of the small intestine: NeuAc alpha 2-3Gal beta 1-4Glc beta 1-Cer (NeuAc-GM3), NeuGc alpha 2-3Gal beta 1-4Glc beta 1-Cer (NeuGc-GM3), GalNAc beta 1-4(NeuGc alpha 2-3)Gal beta 1-4Glc beta 1-Cer (NeuGc-GM2), Gal beta 1-3GalNAc beta 1-4(NeuGc alpha 2-3)Gal beta 1-4Glc beta 1-Cer (NeuGc-GM1), and NeuGc alpha 2-3Gal beta 1-3GalNAc beta 1-4(NeuGc alpha 2-3)Gal beta 1-4Glc beta 1-Cer (NeuGc-GD1a). A positive binding was demonstrated to NeuGc-GM3, NeuGc-GM2, and NeuGc-GD1a, while NeuAc-GM3 and NeuGc-GM1 were negative. The binding pattern differed somewhat for total acid glycolipids of epithelial cells from three different parts of the small intestine. Based on binding preferences of E. coli K99 to a number of glycolipids of various origins, in comparison with calculated minimum energy conformations, a binding epitope was delineated.

Fimbriae were purified from Escherichia coli strains isolated from chickens with septicemia or colibacillosis. When grown on solid media, these strains expressed fimbriae with an apparent subunit molecular mass of 18 kDa. Morphological, biochemical, serological, functional, and molecular characterization revealed that these 18-kDa fimbriae are identical to F11 fimbriae, which were previously found to be involved in the pathogenesis of human urinary tract infection. Screening of a large strain collection showed that 78% of chicken E. coli strains expressed F11 fimbriae, whereas this percentage increased to 96% when the only strains taken into account were those with the serotypes most commonly encountered in avian colibacillosis (O1:K1, O2:K1, O35, and O78:K80). The prevalence of F11 fimbrial expression appeared to be independent of the country of isolation of the strains, except for the United States, where the prevalence seemed higher. Expression of F11 fimbriae by chicken E. coli strains could not be correlated with adherence to chicken tracheal or pharyngeal cells.

The aim of this paper is to describe and discuss relevant aspects of the assessment of physiological functions - and related biomarkers - implicated in the regulation of appetite in humans. A short introduction provides the background and the present state of biomarker research as related to satiety and appetite. The main focus of the paper is on the gastrointestinal tract and its functions and biomarkers related to appetite for which sufficient data are available in human studies. The first section describes how gastric emptying, stomach distension and gut motility influence appetite; the second part describes how selected gastrointestinal peptides are involved in the control of satiety and appetite (ghrelin, cholecystokinin, glucagon-like peptide, peptide tyrosin-tyrosin) and can be used as potential biomarkers. For both sections, methodological aspects (adequacy, accuracy and limitation of the methods) are described. The last section focuses on new developments in techniques and methods for the assessment of physiological targets involved in appetite regulation (including brain imaging, interesting new experimental approaches, targets and markers). The conclusion estimates the relevance of selected biomarkers as representative markers of appetite regulation, in view of the current state of the art.

Summary The bacteriocin release protein (BRP) mediates the secretion of cloacin DF13. The BRP precursor is slowly processed to yield the mature BRP and its stable signal peptide which is also involved in cloacin DF13 secretion. The function of the stable BRP signal peptide was analysed by constructing two plasmids. First, the stable BRP signal peptide was fused to the murein lipoprotein and, second, a stop codon was introduced after the BRP signal sequence. Exchange of the unstable murein lipoprotein signal peptide for the stable BRP signal peptide resulted in an accumulation of precursors of the hybrid murein lipoprotein. This indicated that the BRP signal peptide, as part of this hybrid precursor, is responsible for the slow processing. The stable BRP signal peptide itself was not able to direct the transfer of cloacin DF13 into the periplasmic space or into the culture medium. Over-expression of the BRP signal peptide was lethal and caused ‘lysis’. Subcellular fractionation experiments revealed that the BRP signal peptide is located exclusively in the cytoplasmic membrane whereas the mature BRP, targeted by either the stable BRP signal peptide or the unstable Lpp signal peptide, is located in both the cytoplasmic and outer membrane. These results are in agreement with the hypothesis that the stable signal peptide and the mature BRP together are required for the passage of cloacin DF13 across the cell envelope.

The porcine small intestine contains several polypeptides that could function as receptors for K88-positive Escherichia coli. The mucus fraction contained three proteins with molecular weights of 25, 35 and 60 kDa respectively, which showed a high affinity for K88-positive E. coli cells, whereas brush borders contained a 16 kDa protein and a set of proteins ranging from 40-70 kDa. Depending on the K88 serotype tested, differences in binding to these proteins were observed. In particular, E. coli cells carrying K88ad fimbriae exhibited only a rather weak binding to mucus proteins. The influence of age of the pig on the presence of K88 receptors was also investigated. One-week-old and 35-days-old post-weaning piglets were shown to contain K88 receptors in their mucus while these receptors were hardly detectable in the mucus of 6-month-old pigs. The presence of receptors in the brush border fraction was shown to be independent of age. The binding of K88 fimbriae to mucus proteins was blocked using a lectin of Euonymus europeaus which specifically recognizes the Gal alpha(1-3)Gal sequence, indicating that this disaccharide forms a significant part of the receptor structure.

Specific antisera against FanG and against FanH were prepared by immunization with hybrid Cro-LacZ-FanG and Cro-LacZ-FanH proteins, respectively. Immunoblotting with these antisera revealed the presence of FanG and FanH as minor components in purified K99 fimbriae. Mutations were constructed in fanG and fanH and cells defective in FanG or FanH were characterized by ELISA, immunoblotting, adhesion assays and electron microscopy. A minicell experiment showed that the mutations in fanG or fanH had no effect on the expression of the other K99-specific proteins. Cells defective in FanG produced no fimbriae and did not agglutinate horse erythrocytes, but cell-free heat-shock preparations of these cells still bound the K99 glycolipid receptor. Cells defective in FanH produced 1–2% of the K99 fimbriae as compared with wild-type K99 producing cells. These mutant fimbriae appeared to be shorter but were still capable of binding the K99 glycolipid receptor. Apparently, FanG and FanH are not required for binding the K99 receptor. These results and analysis of K99 mutants by immunoblotting using a specific antiserum against another K99 minor component, FanF, indicated that the combinations FanF FanG and FanF FanH are required for the initiation and elongation (length determination) of K99 fimbriae formation, respectively.

Histochemical profiles were made of identified spinal motoneurons from normal adult zebrafish and from animals subjected to cordotomy or unilateral axotomy of the motor nerves. The lesions caused an increase of the myotomal area with oxidative muscle fibers. We studied the question: do changes in the myotomal muscle configuration concur with changes in the enzyme histochemical profiles of innervating motoneurons? Based on the location and size of cell somata, two categories of motoneurons are distinguished: large white (W) motoneurons that innervate the deep fast, glycolytic muscle fibers, and smaller red and intermediate (RI) motoneurons that innervate the superficial slow oxidative and intermediate muscle fibers. In normal animals, glucose-6-phosphate dehydrogenase activity is high in the large W motoneurons and relatively low in the small RI motoneurons. The reverse holds for succinate dehydrogenase activity is high in the large W motoneurons and relatively low in the small RI motoneurons. The reverse holds for succinate dehydrogenase activity. W and RI motoneurons show similar nicotinamide adenine dinucleotide diaphorase activity. Short- (2 weeks) and long- (8 weeks) term effects of lesions were studied. The results show that: (1) the 3 types of lesions lead to prolonged changes in the enzyme histochemical profiles of spinal motoneurons. The type of change depends on the type of lesion and on the type of motoneuron; (2) unilateral axotomy of the motor nerves affects the histochemical characteristics of spinal motoneurons and the myotomal muscle fiber type configuration on the ipsi- and contralateral side. The contralateral effects are conceived as adaptations to maintain a left-right symmetry in the motor output.

The K88 fimbriae of enterotoxigenic Escherichia coli are strongly immunogenic antigens that can be used to evoke protective immunity. To find out whether these fimbriae can be used as carriers for foreign epitopes, a highly variable region present in the primary structure of the different K88 variants was replaced with five different heterologous epitopes to investigate to what extent these insertions affected the expression, assembly (biogenesis), stability and immunogenic properties of the resulting hybrid fimbriae. Amino acid residues 163–173, were replaced using site-directed in vitro mutagenesis and the hybrid fimbriae were tested for these aspects using ELISA, immunoelectronmicroscopy and immunoblotting. Replacement of this highly variable region did not affect the biosynthesis of fimbriae, although all mutations tested resulted in a reduced expression depending on the epitope inserted. Testing of the different hybrid fimbriae with a panel of monoclonal antibodies raised against the various K88 serotypes K88ab, K88ac and K88ad indicated that replacement of amino acid sequence 163–173 did not affect conserved or K88ab specific epitopes but the K88ac and K88ad specific conformation was lost. Immunization with hybrid fimbriae raises antibodies specific for the inserted heterologous epitopes.

The genetic determinant for the production of 987P fimbriae has been cloned into pBR322. Analysis of frequently occurring deletions in the resultant recombinant plasmid, pPK180, revealed that the 987P gene cluster contains a transposon that encodes the synthesis of heat-stable enterotoxin STpa and is flanked by inverted repeats of IS1. Hybridization experiments with STpa- and 987P-specific probes demonstrated that a variety of STpa+ 987P+ wild-type Escherichia coli strains contained contiguous STpa-987P DNA, most likely on their chromosome. Transcription of the 987P gene cluster appeared to be activated by the adjacent IS1 element.

The influence of temperature on the production of K99 fimbriae by Escherichia coli was determined in cultures growing at constant specific growth rate in continuous cultures. In a wild type strain, in which the K99 operon is present on a low copy number plasmid, low cultivation temperature repressed the K99 production. This temperature-dependent production was not observed after introduction of multicopies of the regulatory region of the K99 operon into this strain, nor in E. coli K12 harbouring a recombinant, multicopy plasmid encoding the K99 operon. These results are in agreement with a regulation model in which a regulatory factor, most likely a repressor, inhibits expression of the K99 operon at low temperatures.

The safety, efficacy and duration of immunity of an improved oil-adjuvant vaccine against haemorrhagic septicaemia, containing inactivated cells of Pasteurella multocida serotype B:2,5, were tested in young buffalo calves in Pakistan. For safety testing, five buffalo calves were vaccinated intramuscularly with twice the normal dose, and six weeks later with a normal dose. Except for a transient rise in rectal temperature at six hours after the vaccinations, no systemic reactions were observed. The buffaloes remained in good condition and had a normal appetite. No local reactions were observed at the injection site. For efficacy testing two trials were carried out. In the first, buffalo calves were vaccinated intramuscularly either with two doses two-and-a-half months apart, or with a single dose, or left unvaccinated. They were challenged subcutaneously with virulent P multocida after eight, 13 or 15 months. After challenge at eight months the four buffaloes given two doses and the buffalo given one dose were protected, whereas the control animal developed the typical signs of the disease. After the challenges at 13 and 15 months, the vaccinated animals were still protected whereas the control animals died. In the second trial, buffalo calves were vaccinated intramuscularly either with two doses two months apart, or with a single dose at two months or left unvaccinated. The buffaloes were challenged after eight or 14 months. After challenge at eight months the four control animals died, whereas three of the four buffaloes given a single dose were protected. After challenge at 14 months, the three control animals died, whereas four of the five buffaloes given two doses and both the buffaloes given a single dose were protected. To test for cross-protection against the heterologous serotypes E:2,5 and B:3,4, groups of mice were vaccinated once or left unvaccinated. Four weeks later, the vaccinated and control groups were challenged with a dilution series of the different challenge cultures. The vaccine appeared to induce protection against challenge with different strains of serotypes B:2,5 and E:2,5 but not against strains of serotype B:3,4.

Children’s habitual diets contain too much high-energy-dense food like lipids and carbohydrates, but little low-energy foods like vegetables. Promoting vegetable intake in children serves better nutritional standards and energy balance. However, since most children dislike vegetables, increasing their intake is a challenge. We investigated the relative effectiveness of repeated exposure (RE) and Flavour-Flavour-Learning (FFL) in increasing vegetable intake and acceptance in pre-schoolers (n = 39). During an intervention period of seven weeks, toddlers consumed vegetable crisps (freeze-dried red beet and parsnip) twice per week. Half of the group received red beet crisps with a dip of tomato-ketchup (C+), and parsnip with a neutral white-sauce (C−), whereas for the other half the order was reversed (red beet C−, parsnip C+). Outcome measures were preference and ad libitum consumption of both crisp flavours (without dip-sauces), measured before, shortly after the intervention, and two and six months following conditioning to assess longer-term effects. Intake increased significantly after the intervention for both vegetables (on average with 8g; an increase of approx. 300%), and this effect was persistent even six months afterwards. The increase was irrespective of crisps being offered with C+ or C− dip sauce. No changes in initial preference for either red beet or parsnip were found. This suggests a robust and persistent effect of RE but no effect of FFL. In conclusion, simply offering pure vegetable tastes repeatedly is sufficient to increase intake over time, rather than adding flavour to the food.

It has been shown that certain fibres can enhance satiety and lower energy intake. This effect largely depends on food matrix, degree of hydration and dosage. Dried chicory root pulp (CRP) is a good fibre-source. After inulin is extracted from chicory roots, the remaining CRP still contains much dietary fibre. We hypothesized that adding CRP to foods may enhance satiety and lower food intake. Moreover, we expected a larger effect when CRP is in a hydrated food (semi-liquid) than in a solid food (granola-bar). We performed a randomized crossover trial with five treatments: a high fibre semi-liquid (14 g CRP), a low fibre semi-liquid (7 g CRP), a control semi-liquid (no CRP), a high fibre bar (14 g CRP), and a control bar (no CRP). Data of 15 lean men (20 ± 3 years) and 15 lean women (21 ± 3 years) were included. Satiety feelings were rated before and multiple times after consumption of the foods. Ninety minutes after consumption, ad libitum energy intake was measured at lunch. High fibre foods enhanced satiety feelings more than their controls (p-values

Pasteurella multocida serotype B:2,5, which causes haemorrhagic septicaemia in buffalo and cattle, was examined for the presence of transferrin-binding proteins. An 82-kDa iron-regulated outer membrane protein was found which specifically binds bovine transferrin. In contrast, P. multocida serotype B:3,4, associated with haemorrhagic septicaemia in feral ruminants, did not express transferrin-binding proteins. These results might indicate a role for transferrin binding in the pathogenesis of haemorrhagic septicaemia in cattle and buffalo.

Summary The interaction of FaeE, a periplasmic chaperone involved in K8B biosynthesis, and the major fimbrial subunit FaeG was Investigated. The genes encoding the two proteins were subcloned together in the expression vector pINIIIA1, Cells expressing the sub-cloned genes accumulated in their periplasm a complex of FaeE and FaeG. This complex was purified by isoelectric focusing and anion-exchange fast-protein liquid chromatography. SOS-PAGE, native gel etectrophoresis, immunoblotting and determination of the N-terminal amino acid sequences and the molar ratio of the W-terminal amino acid residues revealed that the complex is a heterotrimer consisting of two molecules of FaeE and one molecule of FaeG. The periplasmic chaperone FaeE was purified from the periplasm of cells expressing only the subcloned faeE gene. Gel filtration, protein cross-linking analysis and a biophysical approach in which the rotation diffusion coefficient of the purified FaeE was determined led to the conclusion that the native FaeE chaperone is a homodimer.

Spinal motoneurons in the zebrafish were classified using morphological criteria. Dorsomedial white motoneurons which innervate the fast, glycolytic white muscle fiber compartment were distinguished from ventrolateral red and intermediate motoneurons which innervate the slow, oxidative, red and intermediate muscle fiber compartments. Synapses on cell somata and cell organelles were studied in detail. The motoneurons which innervate white muscle fibers (W motoneurons) are considerably larger than those which innervate red and intermediate muscle fibers (RI motoneurons; WRI). Significant differences were also found in the size of the nucleus (WRI) and in the ratio size nucleus/size soma (WRI); small differences were found regarding endoplasmic reticulum (WRI) and mitochondria (WRI). There were no differences in synaptic apposition length or percentage of terminals with flat vesicles. Small differences were discerned with regard to covering percentages (WRI) and percentage of terminals with round vesicles (WRI). Terminals with dense cored vesicles appeared on W motoneuron somata only. Within the motoneuron population, there was a positive correlation between the coverage of terminals containing flat vesicles and the perimeter of the cell soma. In RI motoneurons, there was a positive correlation between the perimeter of the cell and the amount of endoplasmic reticulum. A negative correlation was found between the RI cell perimeter and mitochondria, which is in line with a high succinate dehydrogenase activity in small cells.

Non-acid and acid glycolipids were isolated from the small intestine of a newborn calf and tested for the ability to bind Escherichia coli carrying K99 fimbriae. The bacteria did not bind to any of the non-acid glycolipids, whereas in the acid glycolipid fraction several gangliosides were detected which bind to K99 fimbriae. Gangliosides capable of binding K99 fimbriated E. coli were characterized as NeuGc-GM3, NeuGc-GM2, NeuGc-GD1a NeuAc-SPG and NeuAc-SPG. No binding was detected to NeuAc-GM3 and NeuGc-GM1.

Neonatal and immediate post-weaning diarrhoea in piglets are frequently caused by enterotoxigenic Escherichia coli strains of particular serotypes. Besides producing and excreting enterotoxins these strains possess adhesive antigens which enable the bacteria to adhere to the mucosal surface and colonize the small intestine.1 Well-known adhesive antigens of porcine enterotoxigenic E. coli are the K88, K99 and 987P fimbriae, also designated as fimbrial serotypes F4, F5 and F6, respectively.2

Enzyme histochemical profiles of spinal motoneurons in the zebrafish were determined. Five enzymes of glucose metabolism were chosen: glucose-6-phosphate dehydrogenase (G6PDH), hexokinase (HK), phosphofructokinase (PFK), succinate dehydrogenase (SDH) and NADH tetrazolium reductase (NADH-TR). Motoneurons were traced with Fluorogold and classified as those that innervate white muscle fibres (W-MNs) and those that innervate red and intermediate muscle fibres (R/I-MNs). The average enzyme activities per volume of tissue in the somata of both populations differed at most by 25%. Both the average soma volume and the average number of muscle fibres innervated are three times larger for the W-MNs than for the R/I-MNs. This suggests that the total amount of enzyme activity within a neuron soma matches target size. In the R/I-MNs, the activities of SDH and NADH-TR were closely correlated (correlation coefficient, r = 0.99; p less than 0.05) and HK activity correlated well with G6PDH activity (r = 0.94; p less than 0.05), but not with PFK (r = 0.64; p greater than 0.05). In the W-MNs, there was no correlation between SDH and NADH-TR (r = -0.59; p greater than 0.05) or between HK and G6PDH (r = 0.50; p greater than 0.05) and the correlation coefficient between HK and PFK activity was close to zero (r = 0.04; p greater than 0.05). It was concluded that in the R/I-MNs, which are continuously active, firing activity is fuelled by oxidative metabolism. We suggest that in the W-MNs glucose is stored in the form of glycogen and that, despite high levels of NADH-TR present, the energy for intermittent firing activity is provided by glycolysis.

Summary The nucleotide sequence of faeE and fanE, two genes involved in the biosynthesis of K88 and K99 fimbriae, respectively, was determined and the amino acid sequence of the FaeE and FanE proteins was deduced. Immunobiotting of subcellular fractions with an anti-serum raised against purified FaeE confirmed that FaeE is located in the periplasm. Indications were obtained that FaeE functions as a chaperone-like protein, Its interaction with the fimbrial subunit (FaeG) in the periplasm stabilizes this polypeptide and prevents its degradation by the ceil-envelope protease DegP. Furthermore, FaeE prevents the formation of FaeG multimers which cannot be incorporated into fimbriae. The reactions of the FaeE/FaeG dimers with a set of monoclonal antibodies directed against the various epitopes present on K88 fimbriae revealed that the fimbrial subunits associated with FaeE were present in a conformation resembling their native configuration. Indications about the domains in FaeG involved in the interaction with FaeE are discussed.

The fimbrial adhesins K99 and 987P are produced by enterotoxigenic Escherichia coli strains that provoke neonatal diarrhea in calves and piglets. The biosynthesis of these fimbriae is affected by environmental conditions. Optimal production is achieved at 37°C and high specific growth rate (μ). At lower temperatures or lower specific growth rates the production of fimbriae per unit of biomass rapidly decreases (9). In this study a combination of physiological and genetical approaches was used to elucidate the mechanisms of regulation of fimbriae expression.

This study concerns the identification and location of interneurons in the spinal cord of the zebrafish by way of retrograde tracing from the body musculature. To distinguish motoneurons from interneurons; two tracers were applied: the fluorochrome Fluoro-Gold to label motoneurons, and the trans-synaptically transported compound wheat-germ agglutinin to label motoneurons and the associated interneurons that are considered to be premotoneurons. Because the tracer accumulated mainly in cell bodies (not in neurites), premotoneurons labeled directly from motoneurons could not be distinguished from those labeled via interjacent cells. Both tracers were unilaterally injected into the myotomal muscle at the anal level of the animal. The number of labeled cell bodies in the spinal cord was examined 6, 10 and 16 days after injection. Several technique-oriented experiments were performed in order to map all pathways along with the tracers were incorporated in the neurons. The following observations were made. (1) All Fluoro-Gold-positive cells contained wheat-germ agglutinin, but yet more cells contained only wheat-germ agglutinin; the number of wheat-germ agglutinin-labeled cells was about tenfold higher than the number of Fluoro-Gold-labeled cells. (2) Fluoro-Gold labeling was restricted to cells within one to two spinal cord segments corresponding to the injection site, whereas wheat-germ agglutinin labeling was more diffuse. (3) The position and size of Fluoro-Gold-labeled cells corresponded to those of motoneurons described in previous horse-radish peroxidase experiments. (4) Statistical analysis of the group of wheat-germ agglutinin-labeled cells showed two subpopulations, one with a mean cell size and position corresponding to motoneurons and one with a smaller mean cell size, also positioned within the motor column. The smaller cells were considered to be premotoneurons. The ratio motoneuron:premotoneuron was lowest in the ventrolateral area of the motor column.

Summary The DNA sequence of the K99 fanF gene, encoding FanF, was determined. An open reading frame of 999bp was found. The primary structure of FanF was deduced and analysis revealed the presence of a signal sequence of 22 amino acid residues. The mature protein contains 311 amino acid residues (Mr 33905 D). The amino acid sequence of FanF showed similarity with the K88ab major subunit FaeG. A specific mouse antiserum against FanF was prepared by constructing and purifying a hybrid CroLacZ-FanF protein. Minicell analysis, immunoblotting and immunoelectronmicroscopy revealed a pool of FanF in the periplasm of K99-producing cells and showed, furthermore, that FanF Is a minor component of K99 fibrillae, present at the top and in or along the shaft of the K99 fibrillar structures. A fanF mutant plasmid was constructed. Cells harbouring this plasmid produced all K99-specific proteins, except FanF, but produced 0.1% of the K99 fibrillae relative to ‘normal’ K99-producing cells. Electron microscopic observations showed that cells defective in fanF produce only a few (apparently short) K99 fibrillae. FanF, therefore, was supposed to play a role in initiation and elongation of K99 fibrillae formation. Thin-layer chromatography experiments involving purified receptor material showed that FanF is not required for binding of K99 fibrillae to the ganglioside receptor. Fibrillae produced by an adhesion-negative strain carrying a mutation in the K99 major fibrillar subunit were shown to contain a normal amount of FanF.

So far, three subtypes of the F4 (K88) fimbrial antigen of porcine enterotoxigenic Escherichia coli, F4ab, F4ac, and F4ad, have been distinguished by using polyclonal antisera in agglutination and precipitation tests. The a factor represents one or more common epitopes, whereas each of the b, c, and d factors represents one or more subtype-specific epitopes. We further characterized the F4 antigen complex by using a panel of 40 F4-specific monoclonal antibodies (MAbs). The specificity of all MAbs was proven by enzyme-linked immunosorbent assays, agglutination and radioimmunoprecipitation tests, and immunoelectron microscopy. The MAbs either reacted with all F4 subtypes, reacted with two subtypes, or were subtype specific. Epitope analysis by competition enzyme-linked immunosorbent assays revealed at least 11 epitope clusters on the F4 antigen complex, designated a1 to a7, b1, b2, c, and d. The following antigenic formulas were found for the F4 subtypes: F4ab, a1a2a3a4a5a6b1b2; F4ac, a1a2a3(a4)a5a6a7c; and F4ad, a1a2a3a4a7d. All MAbs were directed against conformational epitopes located on the 27,500-dalton major fimbrial subunits. Consequences for the replacement of polyclonal antisera by MAbs in diagnostic tests are discussed.

SUMMARY: Increase in the production of the fimbrial adhesin K99 by enterotoxigenic Escherichia coli in continuous cultures at specific growth rates above 0·25 h−1was shown to be independent of the nature of the growth-limiting nutrient. The correlation between specific growth rate and K99 production was also found to be independent of the copy number of the K99 operon. Introduction of additional copies of the K99 regulatory region did not affect growth-ratedependent K99 production in wild-type strains, indicating that no hypothetical regulatory host factor is titrated by the K99 regulatory region. Regulation at the transcriptional level was measured with galactokinase gene fusions. The transcription of the fimbrial subunit gene increased with an increase in specific growth rate. This growth-ratedependent transcription was found to originate from the strong promoter PA. Transcription originating from the weaker promoter PB was independent of growth rate. The results indicated that transcriptional regulation at PA is involved in the growth-rate-dependent regulation of K99 production.

We investigated the location and size distribution of motoneurons that innervate red, intermediate and white muscle fibers in the axial musculature of the zebrafish (Brachydanio rerio). Motoneurons were identified by retrograde labeling from the respective myotomal compartments with horseradish peroxidase applied either in small polyacrylamide gel fragments or as pure crystals. We found a spatial relationship between the three myotomal muscle fiber types and the corresponding motoneurons. The white motoneurons are grouped in the dorsal part of the motorcolumn, near the central canal. Motoneurons of the red and intermediate muscle are clustered in the ventral part of the motorcolumn. The average position of the red motoneurons is ventral to that of the intermediate motoneurons. Some sizes are distributed over wide overlapping ranges, spanning from 41 to 352 microns 2 for red and intermediate and from 56 to 894 microns 2 for white motoneurons. These data are discussed in relation to the recruitment order of myotomal muscle fibers of different types as revealed by electromyographic recordings in fish, and the so called "size principle" for motoneuron recruitment.

The nucleotide sequence of the gene encoding the K99 fimbrial subunit of enterotoxigenic Escherichia coli was determined. It appeared that the subunit is composed of 159 amino acid residues preceded by a N-terminal signal sequence of 22 amino acid residues. The secondary structure of the mature K99 polypeptide and the location of potential antigenic determinants were predicted. A comparison was made between the amino acid sequence of the K99 subunit and the subunits of other fimbrial adhesins.

Plasmid pFM205 contains the genetic determinant for the K88ab antigen and is composed of a 4.3-megadalton DNA fragment derived from wild-type K88ab plasmid pRI8801 and cloning vehicle pBR322. The K88 NA of pFM205 contains five genes, which code for polypeptides with apparent molecular weights of 17,000, 26,000 (the K88ab subunit), 27,000 27,500, and 81,000. All five polypeptides were synthesized as precursors approximately 2,000 daltons larger than the mature polypeptides, indicating that they are transported across the cytoplasmic membrane by means of a signal sequence. A set of deletion derivatives of pFM205 was constructed, each containing a deletion in one of the five genes. In strains harboring derivatives of pFM205 containing a deletion in the gene for the 17,000- or 81,000-dalton polypeptide, the K88ab subunit was synthesized and transported to the outside of the cell. However, these strains did not adhere to brushborders or guinea pig erythrocytes, suggesting that the K88ab subunits were not assembled into normal fimbriae. Strains harboring plasmids containing a deletion in the gene for the 27,500-dalton polypeptide still adhered to brush borders and guinea pig erythrocytes, although very little K88ab antigen could be detected with an immunological assay. In strains harboring plasmids containing a deletion in the gene for the 27,000-dalton polypeptide, the K88ab subunit was synthesized but was probably subsequently degraded rapidly.

Treatment of cloacinogenic cultures with increasing concentrations of mitomycin C induced an increasing synthesis of cloacin DF13 accompanied by a decreasing number of colony-forming cells. Cells grown in the presence of glucose required a 10-fold-higher concentration of mitomycin C for optimal induction of cloacin production than did cells grown with lactate. Release of the cloacin was hampered in glucose-grown cells. Experiments with various CloDF13 insertion and deletion mutants revealed that the transcription of CloDF13 deoxyribonucleic acid sequences adjacent to the cloacin structural gene was essential for mitomycin C-induced lethality.

The plasmids CloDF13-clp03 and CloDF13-clp21, obtained after nitrosoguanidine mutagenesis of pCloDF13 (Andreoli, P.M. and Nijkamp, H.J.J. (1976) Mol. Gen. Genet. 144, 159–170), encode mutant bacteriocin molecules with a reduced ability to penetrate susceptible cells (Gaastra, W., Oudega, B. and De Graaf, F.K. (1978) Biochim. Biophys. Acta 540, 301–312). DNA sequence analysis revealed that both the genes encoding the mutant bacteriocin molecules had a point-mutation which resulted in the replacement of proline54 by serine in the amino-terminal domain of the cloacin, involved in translocation. This alteration had no detectable effect on the predicted secondary structure of the proteins or on the interaction with various monoclonal antibodies. Susceptible cells with a relatively low number of receptor proteins were not killed by the bacteriocins or were less susceptible, but Escherichia coli cells with a relatively high number of efficient and functional receptor proteins were efficiently killed. Immunoblotting experiments with the latter type of cells showed that cloacin-clp03, like native cloacin DF13, was fragmented during uptake by the cells, but at a somewhat slower rate.

The cloacin DF13/aerobactin receptor protein from Escherichia coli (pFS8) and from Klebsiella edwardsii were isolated by repeated Triton X-100 extractions and purified by affinity chromatography. Both receptor proteins ran as a single protein band on SDS-PAGE. Their apparent M r values were 74 000 and 76 000, respectively. The binding constants of the purified receptor proteins from E. coli (pFS8) and K. edwardsii and cloacin DF13 were determined. Values of 2.0 × 10 8 M −1 and 1.0 × 10 9 M −1 , respectively, were found. The nucleotide sequence of the pColV-K30 gene, contained on pFS8 and encoding the cloacin DF13/aerobactin receptor protein, was determined and the primary structure of the protein as well as its secondary structure were deduced. The results revealed that the pColV-K30-specified receptor protein might be synthesized as a precursor, with a signal sequence of 25 amino acid residues. The mature protein has an M r of 77 345.

Monoclonal antibodies (MAb) directed against different epitopes on the equimolar complex of cloacin and immunity protein (cloacin DF13) were isolated, characterized, and used to study the uptake of cloacin DF13 by susceptible cells. Four MAbs recognized the amino-terminal part, one MAb recognized the central part, and three MAbs recognized the carboxyl-terminal part of the cloacin molecule. Three MAbs reacted with the immunity protein. Five MAbs inhibited the lethal action of cloacin DF13, but none of the MAbs inhibited the binding of cloacin DF13 to its purified outer membrane receptor protein or the in vitro inactivation of ribosomes. Binding of cloacin DF13 to susceptible cells cultured in broth resulted in a specific, time-dependent dissociation of the complex and a fragmentation of the cloacin molecules. Increasing amounts of immunity protein were detected in the culture medium from about 20 min after the addition of cloacin DF13. Cloacin was fragmented into two carboxyl-terminal fragments with relative molecular masses of 50,000 and 10,000. The larger fragment was detected 5 min after the binding of the bacteriocin complex to the cells. The smaller fragment was detected after 10 min. Both fragments were associated with the cells and could not be detected in the culture supernatant fraction. Cells grown in brain heart infusion were much less susceptible to cloacin DF13 than cells grown in broth, although they possessed a similar number of outer membrane receptor molecules. This decreased susceptibility correlated with a decreased translocation, dissociation, and fragmentation of cloacin DF13.

A plasmid containing a pColV-K30 fragment that encoded only for the cloacin DF13/aerobactin receptor protein was constructed. Escherichia coli cells harboring this plasmid were sensitive to cloacin DF13 but were unable to take up ferric-aerobactin. Another pColV-K30-determined polypeptide (molecular weight, 50,000), localized in the membrane fraction, was essential for the uptake of ferric-aerobactin.

The adhesive antigen K99 of enterotoxigenic Escherichia coli strains of calf origin was isolated and purified. The K99 fimbriae were removed from the cells by heat treatment, concentrated by precipitation with ammonium sulfate, and purified by gel filtration on Sepharose CL-4B and treatment with deoxycholate. The purified K99 antigen was composed of protein subunits with a molecular weight of 18,500 and had an isoelectric point of 9.5. The N-terminal amino acid sequence, as well as the composition of the C-terminal part of the K99 protein subunits, was determined.

A tribrid gene containing ompF, faeC, and lacZ sequences was constructed by subcloning a large central segment of the K88ab gene encoding the fibrillar subunit-like protein FaeC into the open reading frame expression vector pORF2. The resulting tribrid protein was isolated and used to raise antibodies against the FaeC protein. These antibodies were then used for the detection and subcellular localization of the FaeC protein in Escherichia coli harbouring the K88ab-encoding plasmid pFM205 or mutant derivatives. Immunoblotting of subcellular fractions and of purified fibrillae, and agglutination experiments using whole cells revealed that the FaeC protein is present in the periplasm and as a minor component in the K88ab fibrillae. FaeC was also detected in purified K88ac and K88ad fibrillae. Immunoelectron microscopy confirmed the presence of FaeC in K88ab fibrillae, particularly at the tips of the longer fibrillae.