Clonal Relationship among Atypical Enteropathogenic Escherichia coli Strains Isolated from Different Animal Species and Humans

Forty-nine typical and atypical enteropathogenic Escherichia coli (EPEC) strains belonging to different serotypes and isolated from humans, pets (cats and dogs), farm animals (bovines, sheep, and rabbits), and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. A close clonal relationship between human and animal isolates was found by MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out since the transmission dynamics between the reservoirs are not yet clearly understood.Enteropathogenic Escherichia coli (EPEC) strains are among the major causes of infantile diarrhea in developing countries (71) and can be classified as typical and atypical, depending on the presence or absence of the E. coli adherence factor plasmid (pEAF), respectively (39).The pathogenesis of EPEC resides in the ability to cause the attaching and effacing (A/E) lesion in the gut mucosa of human or animal hosts, leading to diarrheal illness (40). The genes responsible for the A/E lesion formation are located in a chromosomal pathogenicity island of ∼35 kb, known as the locus of enterocyte effacement (LEE) (23, 47). LEE encodes an adhesin called intimin (38), its translocated receptor (Tir) (42), components of a type III secretion system (36), and effector molecules, named E. coli-secreted proteins (Esp proteins) (41). These virulence factors have a crucial role in A/E lesion formation, and their detection in EPEC strains is an indicator of their potential to produce these lesions (19, 56).Atypical EPEC strains have been associated with diarrhea outbreaks in developed countries (31, 73, 77) and with sporadic cases of diarrhea in developing and developed countries (1, 12, 26, 52, 55). At present, the prevalence of atypical EPEC is higher than that of typical EPEC in several countries (1, 12, 26, 52, 55, 65).Different from the situation in developed countries, where atypical EPEC outbreaks and sporadic infections are associated with children and adults, atypical EPEC infection in Brazil is mainly associated with children''s illnesses (32, 71).Typical EPEC strains are rarely isolated from animals, and humans are the major natural reservoir for these pathogens (14, 32, 53, 71). In contrast, atypical EPEC strains are present in both healthy and diseased animals (dog, monkey, cats, and bovines) and humans (4, 6, 18, 28, 71). Some studies have associated pets and farm and wild animals as reservoirs and infection sources of atypical EPEC strains for humans (32). However, these studies did not compare atypical EPEC strains isolated from humans and animals by gold-standard molecular methods like multilocus sequence typing (MLST) or pulsed-field gel electrophoresis (PFGE) (15, 35, 43, 53). For this reason, there are some doubts about whether atypical EPEC strains isolated from animals represent risks for human health and whether animals really play the role of reservoirs of atypical EPEC.The aim of this study was to compare atypical EPEC strains isolated from humans and different animals, including pets (cats and dogs), farm animals (bovines, ovines, and rabbits), and wild animals (monkeys), by molecular phylogenetic techniques to verify the role of animals as reservoirs of and sources of infection with atypical EPEC in humans.     

Structural and mutational studies of the catalytic domain of colicin E5: a tRNA-specific ribonuclease

Colicin E5 specifically cleaves four tRNAs in Escherichia coli that contain the modified nucleotide queuosine (Q) at the wobble position, thereby preventing protein synthesis and ultimately resulting in cell death. Here, the crystal structure of the catalytic domain of colicin E5 (E5-CRD) from E. coli was determined at 1.5 A resolution. Unexpectedly, E5-CRD adopts a core folding with a four-stranded beta-sheet packed against an alpha-helix, seen in the well-studied ribonuclease T1 despite a lack of sequence similarity. Beyond the core catalytic domain, an N-terminal helix, a C-terminal beta-strand and loop, and an extended internal loop constitute an RNA binding cleft. Mutational analysis identified five amino acids that were important for tRNA substrate binding and cleavage by E5-CRD. The structure, together with the mutational study, allows us to propose a model of colicin E5-tRNA interactions, suggesting the molecular basis of tRNA substrate recognition and the mechanism of tRNA cleavage by colicin E5.     

T-cell recognition of HLA-DQ2-bound gluten peptides can be influenced by an N-terminal proline at p-1

Recent research has implicated a large number of gluten-derived peptides in the pathogenesis of celiac disease, a preponderantly HLA-DQ2-associated disorder. Current evidence indicates that the core of some of those peptides is ten amino acids long, while HLA class II normally accommodates nine amino acids in the binding groove. We have now investigated this in detail, using gluten-specific T-cell clones, HLA-DQ2-specific peptide-binding assays and molecular modelling. T-cell recognition of both a -gliadin peptide and a low-molecular-weight glutenin peptide was found to be strictly dependent on a ten-amino acids-long peptide. Subsequent peptide-binding studies indicated that the glutenin peptide bound in a conventional p1/p9 register, with an additional proline at p-1. Testing of substitution analogues demonstrated that the nature of the amino acid at p-1 strongly influenced T-cell recognition of the peptide. Moreover, molecular modelling confirmed that the glutenin peptide binds in a p1/p9 register, and that the proline at p-1 points upward towards the T-cell receptor. Database searches indicate that a large number of potential T-cell stimulatory gluten peptides with an additional proline at relative position p-1 exist, suggesting that the recognition of other gluten peptides may depend on this proline as well. This knowledge may be of importance for the identification of additional T-cell stimulatory gluten peptides and the design of a peptide-based, tolerance-inducing therapy.     

An integrated genetic and cytogenetic map for the Mediterranean fruit fly, <Emphasis Type="Italic">Ceratitis capitata</Emphasis>, based on microsatellite and morphological markers

A genetic map based on microsatellite polymorphisms and visible mutations of the Mediterranean fruit fly (medfly), Ceratitis capitata is presented. Genotyping was performed on single flies from several backcross families. The map is composed of 67 microsatellites and 16 visible markers distributed over four linkage groups. Fluorescence in situ hybridization of selected microsatellite markers on salivary gland polytene chromosomes allowed the alignment of these groups to the second, fourth, fifth and sixth chromosome. None of the markers tested showed segregation either with the X or the third chromosome. However, this map constitutes a substantial starting point for a detailed genetic map of C. capitata. The construction of an integrated map covering the whole genome should greatly facilitate genetic studies and future genome sequence projects of the species.     

Dephosphorylation of Neurofilaments by Exogenous Phosphatases Has No Effect on Reassembly of Subunits

Exhaustive in vitro dephosphorylation of porcine neurofilaments (NFs) by alkaline or acid phosphatase did not cause a dissociation of the 210-kD (NF-H), 160-kD (NF-M), or 70-kD (NF-L) subunits and had no effect on the reassembly of NFs from urea or guanidine solution. Electron microscopy revealed that the NFs reassembled from isolated or dephosphorylated subunits had similar morphologies. Phosphatase treatment caused significant increases in the mobilities of NF-M and NF-H on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the subunits underwent marked conformational changes after dephosphorylation. Chemical phosphate analysis showed that as isolated NF-H, NF-M, and NF-L contained about 22, 11, and 3 mol phosphate/mol polypeptide, respectively. The corresponding values for the three subunits from alkaline phosphatase-treated NFs were about 8, 6, and 2 mol phosphate/mol polypeptide, respectively. These results indicate the occurrence of a class of phosphate moieties that is not accessible to exogenous phosphatases.     

Differentiation of cultured human keratinocytes: Effect of culture conditions on lipid composition of normal vs. malignant cells

Summary Differentiation in keratinocytes can be experimentally modulated by changing the culture conditions. When cultured under conventional, submerged conditions, the extent of cellular differentiation is reduced in the presence of low calcium medium and is enhanced in medium containing physiologic calcium concentrations. Moreover cultures grown at the air-medium interface or on a dermal substrate, or both, differentiate even further. Herein we report the effect of culture conditions on lipid composition in normal human keratinocytes and three squamous carcinoma cell (SCC) lines that vary in their capacity to differentiate as assessed by cornified envelope formation. Under submerged conditions, the total phospholipid content was lower, triglyceride content higher, and phospholipid: neutral lipid ratio lower in direct correlation to the degree of differentiation in these cultures. When grown at the air-medium interface on the-epidermized dermis, evidence of further morphologic differentiation was found only for well-differentiated SCC cells and normal keratinocytes. Similarly, the phospholipid content remained high in poorly differentiated SCC cells and it, decreased modestly in well-differentiated SCC cells and markedly in normal keratinocytes. In all cell lines the triglyceride content was increased and cholesterol content decreased when compared to parallel submerged cultures, but these differences were most pronounced in well-differentiated cell lines. Acylceramides and acylglucosylceramides were found only in normal keratinocytes and only under the most differentiation-enhancing conditions. These studies demonstrate differentiation-related changes in the lipid content of both normal and neoplastic keratinocytes. This work was supported in part by NATO Scientific Award (RG 8510056).     

Comparison of the Potency of Competitive NMDA Antagonists Against the Neurotoxicity of Glutamate and NMDA

Abstract: The object of this investigation was to determine whether glutamate uptake affects the apparent potency of the competitive antagonists dl -2-amino-5-phosphonovalerate and CGS-19755 in blocking NMDA receptor-mediated neurotoxicity. In astrocyte-rich rat cortical cultures we observed that dl -2-amino-5-phosphonovalerate and CGS-19755 were 24 and 16 times more potent against NMDA than against glutamate-induced toxicity. In contrast, dl -2-amino-5-phosphonovalerate was equipotent against the two agonists in astrocyte-poor cultures, in which dendrites are directly exposed to the extracellular medium. With the noncompetitive NMDA antagonist MK-801, similar potencies were observed against glutamate (212 ± 16 n M ) and against NMDA (155 ± 9 n M ) neurotoxicity. These results may be explained if we assume that the neuronal cell body is less susceptible than the dendrites to NMDA receptor-mediated toxicity, and that the action of glutamate in astrocyte-rich cultures is confined to the cell body. In this case, one would expect that higher concentrations of glutamate would be needed to produce toxicity in astrocyte-rich cultures, and that higher concentrations of competitive antagonists would be needed to overcome this toxicity. Our observations help explain the pharmacology of the competitive NMDA antagonists against NMDA receptor-mediated neurotoxicity but also suggest the possibility that, because the cell body and dendrites may be distinct sites for neurotoxicity, they might also involve different mechanisms of toxicity.     

Derivatives of Aminoquinones with N-protected Amino Acids

Summary The synthesis of derivatives of aminoquinones with N-protected amino acids is reported here. 2-Amino-1,4-benzoquinone and 2-amino-1,4-naphthoquinone, prepared by the azide method in yields of 60 and 95% respectively, were coupled with N-Boc-protected amino acids including glycine, serine, proline and tyrosine, to give the correspondening derivatives.N, N′-Diisopropylcarbodiimide/1-hydroxybenzotriazole orN, N′-dicyclohexylcarbodiimide/HOBt used as coupling reagents provided the expected products in satisfactory yields and purities as supported by TLC, HPLC and spectral analysis.     

Sister chromatid exchange (SCE) frequencies differ between directly prepared cytotrophoblasts and cultured mesenchymal core cells

Summary Analysis of sister chromatid exchange (SCE) in chorionic villus cells may become useful in measuring the response of fetal tissues to clastogens or mutagens or for prenatal diagnosis of chromosome breakage syndromes such as Bloom syndrome. Previous studies have failed to analyze cytotrophoblastic cells and mesenchymal core cells, or have found no difference between SCE frequencies in directly prepared and cultured cells. Our data indicate significant differences in SCE frequencies between the two cell types: SCE frequency in directly prepared cytotrophoblasts was 6.73 SCE/cell ± 1.6, whereas SCE frequency in cultured mesenchymal core cells was 10.31 SCE/cell ± 0.49 (P < 0.001). SCE analyses involving chorionic villi must take into account cell type.Presented at the 41st Meeting of the American Society of Human Genetics, Cincinnati, Ohio     

Water deprivation increases Fos expression in hypothalamic corticotropin-releasing factor neurons induced by right atrial distension in awake rats

Atrial mechanoreceptors, sensitive to stretch, contribute in regulating heart rate and intravascular volume. The information from those receptors reaches the nucleus tractus solitarius and then the paraventricular nucleus (PVN), known to have a crucial role in the regulation of cardiovascular function. Neurons in the PVN synthesize CRF, AVP, and oxytocin (OT). Stimulation of atrial mechanoreceptors was performed in awake rats implanted with a balloon at the junction of the superior vena cava and right atrium. Plasma ACTH, AVP, and OT concentrations and Fos, CRF, AVP, and OT immunolabeling in the PVN were determined after balloon inflation in hydrated and water-deprived rats. The distension of the balloon increased the plasma ACTH concentrations, which were higher in water-deprived than in hydrated rats (P < 0.05). In addition, the distension in the water-deprived group decreased plasma AVP concentrations (P < 0.05), compared with the respective control group. The distension increased the number of Fos- and double-labeled Fos/CRF neurons in the parvocellular PVN, which was higher in the water-deprived than in the hydrated group (P < 0.01). There was no difference in the Fos expression in magnocellular PVN neurons after distension in hydrated and water-deprived groups, compared with respective controls. In conclusion, parvocellular CRF neurons showed an increase of Fos expression induced by stimulation of right atrial mechanoreceptors, suggesting that CRF participates in the cardiovascular reflex adjustments elicited by volume loading. Activation of CRF neurons in the PVN by cardiovascular reflex is affected by osmotic stimulation.