Isolation of adenoviruses and reoviruses from avian species other than domestic fowl

Adenoviruses and reoviruses were isolated from pigeons and mallard ducks. In addition, adenoviruses were isolated from budgerigars and a bantam and a reovirus was isolated from a turkey. Primary identification of these viruses was by electron-microscope examination. It was further possible to assign the 4 adenoviruses to recognized fowl serotypes, and the reoviruses shared a common antigen with fowl reoviruses. These viruses were isolated from a variety of clinical conditions.     

Characterization of two homologous yeast genes that encode mitochondrial iron transporters

Two different yeast genes were identified that when overexpressed suppressed the low iron growth defect of a mutation in the endoplasmic reticulum iron binding enzyme methyl sterol oxidase. These genes were determined to be novel and highly related. The deduced amino acid sequences indicated that both were membrane proteins having two identical histidine-rich motifs. The predicted proteins, while not ABC transporters, are homologous to a widely distributed family of transition metal transporters present in all kingdoms. Subcellular fractionation and fluorescence microscopy localized these gene products to mitochondria. Based on this result we term these genes Mitochondrial Fe Transporters (MFT). Cells with disruptions in both genes show a growth defect on low iron medium, suggesting that these genes have redundant function and can affect cytosolic iron levels. Measurement of mitochondrial iron in cells grown in iron-rich medium overexpressing MFT1 or MFT2 show a 2-5-fold increase in iron compared with mitochondria from control cells. These results suggest that the mitochondria may act as a reservoir for iron that can be mobilized and used for cytosolic purposes.     

Characterization of two rice MADS box genes that control flowering time

This study utilised positron emission tomography (PET) to identify the cortical areas involved in verbal initiation and suppression in normal subjects whilst performing a sentence completion test (the Hayling Test). In the first condition (response initiation) subjects were required to complete a sentence from which the last word was omitted, whereas in the second condition (response suppression) subjects were asked to complete a sentence with a word which made no sense in the context of the sentence. Subjects were also required to perform a control task in which they had to read out the last word of given sentences. Compared to the control task, response initiation was associated with left-sided activation of the frontal operculum, inferior frontal gyrus, middle temporal gyrus and the right anterior cingulate gyrus, whereas response suppression was associated with left frontal operculum, inferior frontal gyrus and right anterior cingulate gyrus activation. The difference in activation between the two conditions of the Hayling Test lay in the increased activation of the left middle temporal gyrus and the left inferior frontal gyrus during response initiation.     

Characterization of the avian pathogenic Escherichia coli hemagglutinin Tsh, a member of the immunoglobulin A protease-type family of autotransporters

We reported earlier that a single gene, tsh, isolated from a strain of avian pathogenic Escherichia coli (APEC) was sufficient to confer on E. coli K-12 a hemagglutinin-positive phenotype and that the deduced sequence of the Tsh protein shared homology to the serine-type immunoglobulin A (IgA) proteases of Neisseria gonorrhoeae and Haemophilus influenzae. In this report we show that E. coli K-12 containing the recombinant tsh gene produced two proteins, a 106-kDa extracellular protein and a 33-kDa outer membrane protein, and was also able to agglutinate chicken erythrocytes. N-terminal sequence data indicated that the 106-kDa protein, designated Tshs, was derived from the N-terminal end of Tsh after the removal of a 52-amino-acid N-terminal signal peptide, while the 33-kDa protein, designated Tshbeta, was derived from the C-terminal end of Tsh starting at residue N1101. The Tshs domain contains the 7-amino-acid serine protease motif that includes the active-site serine (S259), found also in the secreted domains of the IgA proteases. However, site-directed mutagenesis of S259 did not abolish the hemagglutinin activity or the extracellular secretion of Tshs indicating that host-directed proteolysis was mediating the release of Tshs. Studies with an E. coli K-12 ompT mutant strain showed that the surface protease OmpT was not needed for the secretion of Tshs. Tsh belongs to a subclass of the IgA protease family, which also includes EspC of enteropathogenic E. coli, EspP of enterohemorragic E. coli, and SepA and VirG of Shigella flexneri, which seem to involve a host endopeptidase to achieve extracellular release of their N-terminal domains. In proteolytic studies conducted in vitro, Tshs did not cleave the substrate of the IgA proteases, human IgA1 or chicken IgA, and did not show proteolytic activity in a casein-based assay. Correlation of Tsh expression and hemagglutination activity appears to be a very complex phenomenon, influenced by strain and environmental conditions. Nevertheless, for both APEC and recombinant E. coli K-12 strains containing the tsh gene, it was only the whole bacterial cells and not the cell-free supernatants that could confer hemagglutinin activity. Our results provide insights into the expression, secretion, and proteolytic features of the Tsh protein, which belongs to the growing family of gram-negative bacterial extracellular virulence factors, named autotransporters, which utilize a self-mediated mechanism to achieve export across the bacterial cell envelope.     

A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in redox potential, substrate specificity, and stability

A series of fungal laccases (Polyporus pinsitus, Rhizoctonia solani, Myceliophthora thermophila, Scytalidium thermophilum) and one bilirubin oxidase (Myrothecium verrucaria) have been studied to determine their redox potential, specificity, and stability. Polyporus and Rhizoctonia laccases possess potentials near 0.7-0.8 V (vs. NHE), while other oxidases have potentials near 0.5 V. It is observed that higher redox potential correlates with higher activity. By EPR, no significant change in the geometry of type 1 copper (II) site is observed over this series. At the optimal pH, the two substrates studied, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine, show Km values ranging form 10 to 120 and from 1 to 45 microM; and kcat values ranging from 50 to 16 000 and 200 to 3000 per min, respectively. The enzymes are more stable in the neutral-alkaline pH range. The thermal stability is in the order of bilirubin oxidase equivalent to Myceliophthora laccase equivalent to Scytalidium laccase > Polyporus laccase > Rhizoctonia laccase. Based on these results and the sequence alignments made against Zucchini ascorbate oxidase it is speculated that structural differences in the substrate-activation site (a 'blue', type 1 copper center) control the redox potential range as well as substrate specificity, and the cystine content contributes to stability.     

Growth of solutal dendrites: A cellular automaton model and its quantitative capabilities

A model based on the cellular automaton (CA) technique for the simulation of dendritic growth controlled by solutal effects in the low Péclet number regime was developed. The model does not use an analytical solution to determine the velocity of the solid-liquid (SL) interface as is common in other models, but solves the solute conservation equation subjected to the boundary conditions at the interface. Using this approach, the model does not need to use the concept of marginal stability and stability parameter to uniquely define the steady-state velocity and radius of the dendrite tip. The model indeed contains an expression for the stability parameter, but the process determines its value. The model proposes a solution for the artificial anisotropy in growth kinetics valid at zero and 45° introduced in calculations by the square cells and trapping rules used in previous CA formulations. It also introduces a solution for the calculation of local curvature, which eliminates mesh dependency of calculations. The model is able to reproduce qualitatively most of the dendritic features observed experimentally, such as secondary and tertiary branching, parabolic tip, arms generation, selection and coarsening, etc. Computation results are validated in two ways. First, the simulated secondary dendrite arm spacing (SDAS) is compared with literature values. Then, the predictions of the classic Lipton-Glicksman-Kurz (LGK) theory for steady-state tip velocity are compared with simulated values as a function of melt undercooling. Both comparisons are found to be in very good agreement.     

Synthesis and evaluation of novel rhodacyanine dyes that exhibit antitumor activity

Rhodacyanine dyes and several analogous delocalized lipophilic cations (DLCs) were synthesized and evaluated as novel antitumor agents. Rhodacyanine dye consists of two heteroaromatic rings such as thiazoles at both termini of the conjugate systems and 4-oxothiazolidine (rhodanine) in the middle of it. Compounds with such a unique double-conjugate structure were found to inhibit the growth of several tumor cell lines, such as colon carcinoma CX-1, and to exhibit relatively low toxicity against normal kidney cell line CV-1 (e.g., IC50(CX-1) = 50 nM, IC50(CV-1) = 17.3 microM; selectivity index = 346 for compound 5). These compounds were also found to be efficacious in the tumor-bearing nude mice model (e.g., against human melanoma LOX; T/C (%) = 168 for compound 5). Structural modifications on rhodacyanine, including deletion of a heteroaromatic ring involved in the merocyanine conjugate system and replacement of rhodanine with a structurally related moiety such as 4-oxoimidazolidine or 4-oxo-1,3-dithiolane, resulted in a loss of the selectivity and/or the activity. Our current structure-activity studies imply that the double-conjugate system with a rhodanine moiety is essential for the selective activity of rhodacyanine dyes, and we find this class of compounds as unique antitumor agents candidates.     

Characterization of two second messenger pathways and their interactions in eliciting the human sperm acrosome reaction

Actinobacillus actinomycetemcomitans, a gram-negative, capnophilic bacterium, is associated with several human diseases and is the suspected etiologic agent in certain forms of periodontal disease. We have previously shown that this organism produces an immunosuppressive factor (ISF) which is capable of inhibiting both T- and B-cell activation. Furthermore, these effects appear to be associated with the activation of a population of suppressor cells. We now report that the ISF induces a unique population of CD4+CD8+ dual-positive T-cells. By utilizing multiparameter flow cytometric analysis, we were able to detect the presence of dual-positive cells in cultures of human T-cells treated with PHA and ISF. The cells appeared within 48 hr and their induction was dependent upon the presence of both CD4 and CD8 cells in the culture. Dual expression of CD4 and CD8 was stable in that the cells continued to express both surface proteins after being sorted and cultured for an additional 24 hr. Phenotypic analysis indicates that these cells are also CD3+, CD2+, CD5+, TCR alpha beta+, CD45RA+ (and RO+), and CD29+. The dual-positive cells express surface markers associated with T-cell activation: CD25+, CD69+, CD71+, and HLA-DR+. In contrast, the cells were negative for CD34, CD57, CD56, and CD16. Cell cycle analysis indicates that > 80% of the dual-positive cells were in the S phase. Finally, functional analysis of these cells indicates that they are capable of suppressing the proliferative response of autologous T-cells to PHA.     

Smooth muscle cells isolated from discrete compartments of the mature vascular media exhibit unique phenotypes and distinct growth capabilities

Heterogeneity of smooth muscle cell (SMC) phenotype and function is rapidly emerging as an important concept. We have recently described that phenotypically distinct SMC subpopulations in bovine pulmonary arteries exhibit unique proliferative and matrix-producing responses to hypoxic pulmonary hypertension. To provide better understanding of the molecular mechanisms contributing to this phenomenon, experimental studies will require a reliable in vitro model. The purpose of the present study was first to determine if distinct SMC subpopulations, similar to those observed in vivo, could be selectively isolated from the mature arterial media, and then to evaluate whether select SMC subpopulations would exhibit heightened responses to growth-promoting stimuli and hypoxia. We were able to reproducibly isolate at least four phenotypically unique cell subpopulations from the inner, middle, and outer compartments of the arterial media. Differences in cell phenotype were demonstrated by morphological appearance and differential expression of muscle-specific proteins. The isolated cell subpopulations exhibited markedly different growth capabilities. Two SMC subpopulations grew slowly in 10% serum and were quiescent in plasma-based medium. The other two cell subpopulations, exhibiting nonmuscle characteristics, grew rapidly in 10% serum and proliferated in plasma-based medium and in response to hypoxia. Certain colonies of the nonmuscle-like cell subpopulations were found to grow autonomously under serum-deprived conditions and to secrete mitogenic factors. Our data, demonstrating that phenotypically distinct cells with enhanced growth potential exist within the normal arterial media, support the idea that these unique cells could contribute selectively to the pathogenesis of vascular disease.     

Reconcilable differences: the marriage of qualitative and quantitative methods

Qualitative research consists of methods that allow for a more in-depth understanding of phenomena and encompasses techniques such as focus groups, in-depth interviews, and participant observation. The guidelines that pertain to sampling and analysis are different from those which govern quantitative techniques, but they can be applied just as rigorously to ensure the validity of the results. This article introduces these methods and criteria and illustrates how qualitative and quantitative methods can be combined in order to improve what is learned from each.     

Ultrastructural differences induced by heat in red and white avian muscles in rigor mortis

A species difference in the intercellular adhesive selectivity of mixtures of embryonic liver cells is reported. This is first quantitative assessment of species differences in the intercellular adhesive properties of embryonic cells. A collecting aggregate assay, a new double-label assay procedure, and histological and autoradiographic procedures were used to elucidate the intercellular adhesive selectivity of developing mammalian and avian liver cells. Evidence is presented that the reported adhesive differences are not due to the different cell types composing the respective embryonic mammalian and avian livers. Finally, such heterolgous-homotypic selectivity of adhesion is not a property of all tissues, since it is shown that developing brain cells (mesencephalon) do not exhibit the avove intercellular adhesive selectivity (mammalian vs. avian). These findings provide further support for the hypothesis that generic identity as well as cell type may play an important part in determining the intercellular adhesive behavior of heterologous-homotypic mixtures of embryonic cells. A possible evolutionary divergence of morphogenetic mechanisms is discussed.     

Characterization of outward currents in neurons of the avian nucleus magnocellularis

Characterization of outward currents in neurons of the avian nucleus magnocellularis. J. Neurophysiol. 80: 2824-2835, 1998. Neurons of the nucleus magnocellularis (NM) preserve the timing of auditory signals through the convergence of a variety of voltage- and ligand-gated ion channels. To understand better how these channels interact, we have characterized the kinetics, voltage sensitivity, and pharmacology of outward currents of NM neurons in brain slices. The reversal potential (Erev) of outward currents varied with potassium concentration as expected for currents carried by potassium. However, Erev was consistently more positive than the Nernst potential for potassium (EK). Deviation of Erev from the calculated EK most likely arose from potassium accumulation in extracellular spaces by potassium conductances active at rest and during depolarizing steps. Three outward potassium currents were studied that varied in voltage and pharmacological sensitivity. A tetraethylammonium (TEA)-sensitive, high-threshold current was activated within 1-5 ms of the onset of depolarization, with a half-maximal activation voltage (V1/2) of -19 mV. It was blocked partially by 4-aminopyridine (4-AP) and was the dominant ionic conductance of NM neurons. A dendrotoxin-I (DTX) and 4-AP-sensitive, low-threshold current had a V1/2 of -58 mV, rapid activation kinetics, and only partial inactivation, with decay time constants between 20 and 100 ms. A rapidly inactivating current was observed that was resistant to TEA and DTX and was blocked by intracellular Cs+. The transient current was inactivated almost completely at the resting potential. The onset of inactivation was fastest at potentials negative to those that caused activation. When intracellular K+ was replaced by Cs+, large inward and outward currents were obtained that corresponded respectively to the above-mentioned DTX- and TEA-sensitive currents. Outward, TEA-sensitive current was carried by Cs+, with a PCs/PK of approximately 0.1. In current-clamped neurons, DTX induced repetitive firing and increased membrane time constant near rest but had little effect on action potential duration. These studies indicate that a low-threshold, DTX-sensitive current plays a key role in making NM neurons highly responsive to the onset and offset of synaptic stimuli.     

Colonization ability and pathogenic properties of a fim- mutant of an avian strain of Escherichia coli

Several studies suggest that the expression of type 1 fimbriae is involved in the virulence of Escherichia coli in chickens, by promoting adhesion of bacteria to the respiratory tract, which is most probably the first step to occur in the infection, and by interacting with the immune response. In order to determine to what extent type 1 fimbriae were involved in the pathogenic process, the fim cluster of an avian pathogenic strain of E. coli, MT78 (O2:K1:H+), was modified in vitro and reintroduced in the parent strain via allele exchange using suicide vector pCVD442. The mutant strain thus generated (DM34) had its entire fim cluster removed. Its pathogenic properties were compared to those of the parent strain in an experimental reproduction of avain colibacillosis in 15-day-old chickens, after primary infection with infectious bronchitis virus followed by intratracheal inoculation of the challenge strain. In specific-pathogen-free (SPF) animals, mutant DM34 was less pathogenic than the parent strain and colonized the lungs of infected animals to a lower level. In germ-free chickens, although DM34 was less pathogenic than MT78 according to the differences in weight gains, it colonized the trachea, lungs and internal organs to the same extent as MT78. Our results suggest that, whereas type 1 fimbriae are not strictly required in colonization of the respiratory tract of germ-free chickens, they might be important in establishing a bacterial population in the lungs of SPF animals. The difference regularly observed in weight gains between mutant- and wild-type-inoculated chickens reflects a decreased pathogenicity of the fim- mutant. However, the isolation of E. coli in target organs and the observation of colibacillosis symptoms and lesions in mutant-inoculated chickens suggest that type 1 fimbriae by themselves play a limited role in pathogenicity.     

Conditionally pathogenic mycobacteria in the environment and their effect on living organisms

Since 1968 when bovine tuberculosis was eliminated in the Czech Republic, the epidemiological situation of bovine tuberculosis has been stabilized. At present the incidence of the disease in men and animals caused by conditionally pathogenic mycobacteria is worldwide increasing. In human population, especially people with impaired immunity are affected. In farm animals infections caused by conditionally pathogenic mycobacteria may often result in complications in intravital and postmortal diagnosis of bovine and avian tuberculosis. Those infections are then often incorrectly diagnosed which could have a great negative impact on health, economy and breeding. Therefore the objective of the study was to summarize data from literature and our own experience concerning the occurrence of atypical mycobacteria in environment. The study is divided into 5 summarizing chapters, supplemented with 13 Tables.     

Differential pathogenic effect of two Helicobacter-like organisms in dog gastric mucosa

Postorchidectomy treatment options in patients with stage I seminoma include surveillance (reserving treatment for patients who relapse), adjuvant radiation therapy (RT), and adjuvant chemotherapy. Adjuvant retroperitoneal RT remains the treatment of choice in most centers; however, the success of surveillance in stage I nonseminomatous germ cell testis tumors, the establishment of curative chemotherapy for advanced disease, and the improvements in CT have led to re-examination of the standard treatment approach. The available data from the surveillance and adjuvant RT series suggest that almost 100% of patients with stage I testicular seminoma are cured, whichever approach is chosen. This article presents an overview of the available information on all treatment options, the pros and cons of each approach, and indications for where surveillance fits into the armamentarium of clinicians dealing with this disease.