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PubMed1986

Polyamine depletion following exposure to DL-alpha-difluoromethylornithine both in vivo and in vitro initiates morphological alterations and mitochondrial activation in a monomorphic strain of Trypanosoma brucei brucei.

DL-alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase (ODC), rapidly depletes cells of intracellular putrescine. When administered to animals and humans, DFMO cures acute infections of trypanosomiasis. In order to determine if the mechanism of drug action is related to initiation of transformation and biochemical alterations subsequent to polyamine depletion, trypanosome morphology and mitochondrial activation were studied in a monomorphic strain of Trypanosoma brucei brucei. Exposure of trypanosomes to DFMO in vivo in infected rodents or in vitro in culture resulted in a depletion of intracellular putrescine and a cessation of cell division without specific cytotoxicity. These events were followed by a transformation of the long slender bloodstream form to a short stumpy form via an intermediate morphology. Putrescine, the product of the ODC reaction, abrogates this effect. When introduced into SDM-79 medium, the intermediate form is capable of further transformation to an "insect" procyclic trypomastigote whereas the long slender form and short stumpy form are not. Short stumpy forms are incapable of binary fission and have lost their infectivity for the vertebrate host. In addition, the mitochondrial marker enzyme, NAD diaphorase, was found only in the short stumpy and intermediate forms. We hypothesize that the short stumpy phenotype may not be a viable stage in the natural transformation of the trypanosome from its mammalian host to the insect vector.

Animals↗
PubMed1990

Calculation of site affinity constants and cooperativity coefficients for binding of ligands and/or protons to macromolecules. II. Relationships between chemical model and partition function algorithm.

The relationships between the chemical properties of a system and the partition function algorithm as applied to the description of multiple equilibria in solution are explained. The partition functions ZM, ZA, and ZH are obtained from powers of the binary generating functions Jj = (1 + kappa j gamma j,i[Y])i tau j, where i tau j = p tau j, q tau j, or r tau j represent the maximum number of sites in sites in class j, for Y = M, A, or H, respectively. Each term of the generating function can be considered an element (ij) of a vector Jj and each power of the cooperativity factor gamma ij,i can be considered an element of a diagonal cooperativity matrix gamma j. The vectors Jj are combined in tensor product matrices L tau = (J1) [J2]...[Jj]..., thus representing different receptor-ligand combinations. The partition functions are obtained by summing elements of the tensor matrices. The relationship of the partition functions with the total chemical amounts TM, TA, and TH has been found. The aim is to describe the total chemical amounts TM, TA, and TH as functions of the site affinity constants kappa j and cooperativity coefficients bj. The total amounts are calculated from the sum of elements of tensor matrices Ll. Each set of indices (pj..., qj..., rj...) represents one element of a tensor matrix L tau and defines each term of the summation. Each term corresponds to the concentration of a chemical microspecies. The distinction between microspecies MpjAqjHrj with ligands bound on specific sites and macrospecies MpAqHR corresponding to a chemical stoichiometric composition is shown. The translation of the properties of chemical model schemes into the algorithms for the generation of partition functions is illustrated with reference to a series of examples of gradually increasing complexity. The equilibria examined concern: (1) a unique class of sites; (2) the protonation of a base with two classes of sites; (3) the simultaneous binding of ligand A and proton H to a macromolecule or receptor M with four classes of sites; and (4) the binding to a macromolecule M of ligand A which is in turn a receptor for proton H. With reference to a specific example, it is shown how a computer program for least-squares refinement of variables kappa j and bj can be organized. The chemical model from the free components M, A, and H to the saturated macrospecies MpAQHR, with possible complex macrospecies MpAq and AHR, is defined first.(ABSTRACT TRUNCATED AT 250 WORDS)

Algorithms↗
PubMed1985

Differentiation in Trypanosoma brucei: host-parasite cell junctions and their persistence during acquisition of the variable antigen coat.

Acquisition of the variable antigen-containing surface coat of Trypanosoma brucei occurs at the metacyclic stage in the salivary glands of the tsetse fly vector. The differentiation of the metacyclic trypanosome in the gland has been studied by scanning electron microscopy and by transmission electron microscopy of thin sections and freeze-fracture replicas. The uncoated epimastigote trypanosomes (with a prenuclear kinetoplast) divide while attached to the salivary gland epithelium brush border by elaborate branched flagellar outgrowths, which ramify between the host cell microvilli and form punctate hemidesmosome-like attachment plaques where they are indented by the microvilli. These outgrowths become reduced as the epimastigotes transform to uncoated trypomastigotes (with postnuclear kinetoplast), which remain attached and capable of binary fission. The flagellar outgrowths disappear but the attachment plaques persist as the uncoated trypomastigotes (premetacyclics) stop dividing and acquire the surface coat to become 'nascent metacyclics'. Coat acquisition therefore occurs in the attached trypanosome and not, as previously believed, after detachment. Coating is accompanied by morphological changes in the glycosomes and mitochondrion of the parasite. Freeze-fracture replicas of the host-parasite junctional complexes show membrane particle aggregates on the host membrane but not on the parasite membrane. It is suggested that disruption of the complex occurs when maximum packing of the glycoprotein molecules has been achieved in the trypanosome surface coat, releasing the metacyclic trypanosome into the lumen of the gland.

Animals↗
PubMed1987

Cloning of the genes involved in synthesis of coenzyme pyrrolo-quinoline-quinone from Acinetobacter calcoaceticus.

Mutants of Acinetobacter calcoaceticus LMD79.41 were isolated that are defective in the synthesis of the coenzyme pyrrolo-quinoline-quinone (PQQ). A gene bank of the wild-type. A. calcoaceticus genome was constructed with the binary plasmid system pLV21-RP4 delta Km. The DNA of A. calcoaceticus LMD79.41 was partially digested with Sau3A, and fragments of about 15 kilobases were inserted into the BamHI site of pLV21. The hybrid plasmids maintained in Escherichia coli were transferred by conjugation to the PQQ- mutants of A. calcoaceticus. One hybrid plasmid was isolated that complements all isolated PQQ- mutants. Subcloning of this plasmid in the vector pRK290 resulted in an insert of 5 kilobases on which at least four different genes involved in PQQ synthesis could be indicated. With Tn5 insertions the four PQQ genes were mapped, and it was shown that these genes are most probably located in three operons.

Acinetobacter↗
PubMedDate not supplied

Hybrid formation between African trypanosomes during cyclical transmission.

Trypanosomes of the species Trypanosoma brucei reproduce primarily by binary fission, but the frequency of enzyme electrophoretic variants in natural populations of T. brucei has provided indirect evidence for the existence of a sexual cycle. These studies, coupled with studies of restriction fragment length polymorphisms of genes encoding glycolytic enzymes, have also provided evidence for T. brucei being diploid. Here we report direct evidence of gene exchange between two different clones of trypanosomes after mixed infection and full cyclical development in the tsetse fly vector.

Animals↗
PubMed1990

Dissecting the domain structure of the regulatory subunit of cAMP-dependent protein kinase I and elucidating the role of MgATP.

A truncated regulatory subunit of cAMP-dependent protein kinase I was constructed which contained deletions at both the carboxyl terminus and at the amino terminus. The entire carboxyl-terminal cAMP-binding domain was deleted as well as the first 92 residues up to the hinge region. This monomeric truncated protein still forms a complex with the catalytic subunit, and activation of this complex is mediated by cAMP. The affinity of this mutant holoenzyme for cAMP and its activation by cAMP are nearly identical to holoenzyme formed with a regulatory subunit having only the carboxyl-terminal deletion and very similar to native holoenzyme. The off rate for cAMP from both mutant regulatory subunits, however, is monophasic and very fast relative to the biphasic off rate seen for the native regulatory subunit. The effects of NaCl, urea, and pH on cAMP binding are also very similar for the mutant and native holoenzymes. Like the native type I holoenzyme, both mutant holoenzymes bind ATP with a high affinity. The positive cooperativity seen for MgATP binding to the native holoenzyme, however, is abolished in the double deletion mutant. The Hill coefficient for ATP binding to this mutant holoenzyme is 1.0 in contrast to 1.6 for the native holoenzyme. The Kd (cAMP) is increased by approximately 1 order of magnitude for both mutant forms of the holoenzyme in the presence of MgATP. A similar shift is seen for the native holoenzyme. Further characterization of the MgATP-binding properties of the wild-type holoenzyme indicates that a binary complex containing catalytic subunit and MgATP is required, in particular, for reassociation with the cAMP-bound regulatory subunit. This binary complex is required for rapid dissociation of the bound cAMP and is probably responsible for the observed reduction in cAMP-binding affinity for the type I holoenzyme in the presence of MgATP.

Adenosine Triphosphate↗
PubMed1987

Demonstration of colonies of Cowdria ruminantium in midgut epithelial cells of Amblyomma variegatum.

The development of colonies of Cowdria ruminantium was studied in midgut epithelial cells of adult Amblyomma variegatum that had become infected by feeding as nymphs on cattle with experimentally induced heart-water disease. Colonies were not observed in gut tissues obtained from nymphs during the feeding period, but were present in midgut epithelial cells of ticks obtained at 15 days after they were replete through molting to the adult stage. Colonies were small (1 to 10 micron) initially, but as tick development progressed, their diameter increased to as much as 60 micron. With electron microscopy, colonies were observed to be membrane bound and contained pleomorphic organisms that were reticulated. The organisms seemed to be dividing by binary fission. Many colonies contained a large, electron-dense inclusion that was morphologically similar to hemoglobin deposits found in the cytoplasm of midgut epithelial cells of recently fed ticks. Cowdria ruminantium was often observed adhered to these inclusions.

Animals↗
PubMed1979

Babesia microti: morphology, distribution and host relationship in Germany.

A short review has been given about recent studies on Babesia microti in natural animal host's near Munich (Southern Germany). An infected area has been studied in order to elucidate the outdoor relationship between the local strains and their preferred hosts, the European field vole Microtus agrestis. The seasonal variation of the parasites prevalence in voles shows a rise in the early summertime (71% of the catches infected). Roundish forms predominate in the erythrocytes. Multiplication never takes place by binary fission, but in "Maltese cross" form or budding-like. Normally the parasite is enveloped with a simple elementary membrane only, as shown by electron microscopy. The arthropod host is still not identified, infections of human beings not observed. This is the first finding and full description of B. microti in Germany.

Animals↗
PubMed1984

Stabilized binary hierarchic classifier in cytopathologic diagnosis.

A binary tree classifier (BTC) algorithm for computer-assisted cell image analysis has been developed that overcomes the problem of overtraining due to inadequate sample size/dimensionality ratio at the higher-order nodes of a hierarchic decision structure. Provisions have been introduced that ensure that decision rules created at each node are based on samples representative of the subpopulation routed to the node. These provisions eliminate problems caused by truncation effects resulting from the application of decision rules at preceding decision nodes. The BTC performs better than do single-stage classifiers in situations where the categories' mean vectors are not well separated and no equality of covariance matrices exists. In applications in which noticeable deterioration of classifier performance on test-set data is common, the classification success rate of the BTC algorithm is not statistically significantly different between the training-set and test-set data.

Cells↗
PubMed1989

Dependence on pH of substrate binding to a mutant lactose carrier, lacYun, in Escherichia coli. A model for H+/lactose symport.

The lacYun gene, which encodes a lactose carrier showing the uncoupled phenotype of substrate transport in Escherichia coli [Wilson, Kusch & Kashket (1970) Biochem. Biophys. Res. Commun. 40, 1409-1414], was cloned on a plasmid vector, pBR322. The binding of a substrate, p-nitrophenyl alpha-galactoside, to the lacYun carrier in membranes from the strain harbouring the lacYun clone showed a pH-dependence different from its binding to the wild-type lactose carrier. This finding indicated that the lacYun mutation confers higher affinity for H+ on the carrier, exerting its effect on the less efficient dissociation of substrate inside cells. The result coincides with the proposal [Yamato & Rosenbusch (1983) FEBS Lett. 151, 102-104] that the proton affecting the substrate binding is the coupling proton of the proton/lactose symport reaction, which allows only the ordered mechanism of binding of substrate to an H+-carrier binary complex. From the simplest model of the symport reaction, constructed on the basis of these results, the coupling site of energy in the carrier cycle of the transport reaction can be identified at the substrate-dissociation step inside cells.

Binding Sites↗
PubMed1991

Comparative study on Rickettsia-like organisms in the midgut epithelial cells of different Glossina species.

The midgut epithelium of Glossina morsitans centralis, G. austeni, G. pallidipes, G. palpalis palpalis, G. p. gambiensis, G. fuscipes fuscipes, G. tachinoides and G. brevipalpis from ILRAD-bred colonies was examined, by electron microscopy, for the presence and distribution of Rickettsia-like organisms (RLOs). RLOs were present in the midgut epithelial cells of all non-teneral tsetse. In G.m. centralis, G. pallidipes and, to a much lesser extent, G. brevipalpis, RLOs were numerous and were present in all the specimens examined. RLOs were present in fewer numbers in the epithelial cells of tenerals of these three tsetse species. In contrast, RLOs occurred in very much lower numbers within the midgut cells of nonteneral G. austeni, G. p. palpalis, G. p. gambiensis, G.f. fuscipes and G. tachinoides; were not seen in every specimen, and were rarely observed in the midgut cells of teneral testse. The RLOs were typical rod-shaped bacteria with an inner and outer membrane, which occurred free within the host cell cytoplasm and appeared to cause no obvious pathology. The micro-organisms divided by binary fission and at least two distinct morphological forms plus a range of intermediate forms were seen in the midgut cells. A comparison of the presence and numbers of RLOs within the midgut cells and the midgut infection rates of both Trypanosoma congolense and T. b. brucei, both between Glossina species and also within the same stock of tsetse, clearly indicates that the ability of trypanosomes to establish and develop to mature infections is unlikely to be correlated solely with the presence of RLOs within the tsetse midgut.

Animals↗
PubMed2025

Disease candidate genes prediction using positive labeled and unlabeled instances.

Identifying disease genes and understanding their performance is critical in producing drugs for genetic diseases. Nowadays, laboratory approaches are not only used for disease gene identification but also using computational approaches like machine learning are becoming considerable for this purpose. In machine learning methods, researchers can only use two data types (disease genes and unknown genes) to predict disease candidate genes. Notably, there is no source for the negative data set. The proposed method is a two-step process: The first step is the extraction of reliable negative genes from a set of unlabeled genes by one-class learning and a filter based on distance indicators from known disease genes; this step is performed separately for each disease. The second step is the learning of a binary model using causing genes of each disease as a positive learning set and the reliable negative genes extracted from that disease. Each gene in the unlabeled gene's production and ranking step is assigned a normalized score using two filters and a learned model. Consequently, disease genes are predicted and ranked. The proposed method evaluation of various six diseases and Cancer class indicates better results than other studies.

Humans↗
PubMed1991

Expression of v-src in embryonic neural retina alters cell adhesion, inhibits histogenesis, and prevents induction of glutamine synthetase.

Using Rous sarcoma virus as the vector, v-src or c-src genes were introduced into 6-day chicken embryo retina tissue in organ culture and their effects on retina development were investigated. Overexpression of c-src in many of the cells had no noticeable effect on retina development. In contrast, infection with v-src resulted in abnormal histogenesis and inhibition of differentiation. Although only a portion of the cells in infected tissue expressed the oncogene and displayed the transformation phenotype, the other cells were also hindered from becoming normally positioned and organized. Therefore, presence of oncogene-transformed cells within the tissue hindered organization and development of adjacent nontransformed cells. Failure of normal cell relationships impeded induction by cortisol of glutamine synthetase in Muller glia, which requires contact associations of the glia cells with neurons. The transformed cells tended to assemble into chaotic clusters, suggesting that their adhesiveness and contact affinities had become altered. This was confirmed by aggregation experiments with dissociated cells which showed that adhesiveness of transformed cells was greatly reduced and that they had lost the ability to cohere with nontransformed cells. In binary mixtures of transformed and nontransformed cells, the two sorted out into separate aggregates. Transformed cells formed loose clusters devoid of tissue architecture; aggregates of nontransformed cells became organized into retinotypic structures, and glutamine synthetase was inducible. Our findings suggest that the mechanisms of cell adhesion and cell affinities are a key target of v-src activity in infected cells and that modification of the cell surface may be a leading factor in other cellular changes characteristic of the v-src transformation phenotype.

Animals↗
PubMed1987

Morphology and development of Cowdria ruminantium in Amblyomma ticks.

The morphology and development of Cowdria ruminantium have been studied in Amblyomma hebraeum and A. variegatum. Colonies of C. ruminantium have so far been demonstrated microscopically in gut, salivary gland cells, haemocytes and malphighian tubules of infected Amblyomma ticks. Colonies in gut cells were seen in both unfed and feeding ticks but colonies in salivary gland acini were observed only in nymphs that had fed for 4 days. Although the predominant type seen in both tick stages was the reticulated form that appeared to divide by binary fission, electron dense forms were also present. The latter are similar to those forms documented in endothelial cells of the vertebrate host as well as in cell culture. The presence of colonies of C. ruminantium in salivary glands of feeding ticks, along with the demonstration of different morphologic forms of the organism, suggests that a developmental cycle of the organism occurs in its invertebrate host. It is thought that organisms first infect and develop within gut cells. From there subsequent stages continue their development in haemolymph and salivary glands and are then transferred to the vertebrate host during tick feeding. Further studies are needed to completely understand the development of C. ruminantium in ticks and its subsequent transmission by these parasites.

Animals↗
PubMed1991

Requirements for cell surface expression of the human TCR/CD3 complex in non-T cells.

The T-cell antigen receptor (TCR) consists of a glycoprotein heterodimer (alpha/beta or gamma/delta) which is non-covalently associated with at least four or five invariant polypeptides (CD3 gamma, delta, epsilon, zeta and eta). In T-cell variants lacking TCR alpha, beta or zeta, it has been shown that incomplete TCR/CD3 complexes are retained within the cell. To examine requirements for cell surface expression of TCR/CD3, we transfected COS monkey kidney cells with cDNAs encoding TCR alpha, beta and CD3 gamma, delta, epsilon and zeta. We report that cell surface appearance of TCR/CD3 on COS cells requires coordinate expression of all six proteins. In the absence of the zeta chain, subcomplexes comprising from two to five chains were readily demonstrable in COS cells, but they failed to reach the cell surface or to acquire N-linked oligosaccharide side chains indicating failure to reach the medial Golgi. Pulse-chase metabolic labelling of transfected COS cells showed that three chains (CD3 gamma, CD3 epsilon, and zeta) were stable while three (TCR alpha, TCR beta and CD3 delta) were rapidly degraded. In two- and three-chain co-transfections specific intracellular subcomplexes were formed between TCR alpha and CD3 gamma, TCR alpha and CD3 delta, or TCR beta and CD3 epsilon. Binary subcomplexes having at least one stable chain (CD3 epsilon - TCR beta) were stable while one formed by two unstable chains (TCR alpha - CD3 delta) was still degraded. Assembly of the TCR/CD3 complex in COS cells thus appears centered around the metabolically stable CD3 gamma and CD3 epsilon proteins. Site-specific mutations of the negatively-charged transmembrane amino acid of residues of the CD3 chains to alanines served to either abolish (for TCR alpha - CD3 delta and TCR beta - CD3 epsilon) or diminish (for TCR alpha -CD3 gamma) these TCR-CD3 interactions. These mutations had no effect, however, on CD3-CD3 interactions or upon synthesis, metabolism, or intracellular distributions of the CD3 proteins. The transmembrane domains of CD3 gamma, delta, and epsilon thus appear to play a major role in associations of CD3 with TCR chains.

Animals↗
PubMed1991

Binary system for regulating transgene expression in mice: targeting int-2 gene expression with yeast GAL4/UAS control elements.

We have developed a binary transgenic system that activates an otherwise silent transgene in the progeny of a simple genetic cross. The system consists of two types of transgenic mouse strains, targets and transactivators. A target strain bears a transgene controlled by yeast regulatory sequences (UAS) that respond only to the yeast transcriptional activator GAL4. A transactivator strain expresses an active GAL4 gene that can be driven by any selected promoter. The current paradigm uses the murine growth factor int-2 cDNA as the target gene and the GAL4 gene driven by the mouse mammary tumor virus long terminal repeat as the transactivator. Both target and transactivator strains are phenotypically normal. By contrast, the bigenic offspring of these two strains express high levels of the target int-2 gene in each organ expressing the GAL4 transactivator. They also display a characteristic dominant int-2 phenotype that consists of epithelial hyperplasia in mammary and salivary glands, as well as prostatic and epididymal hypertrophy, which results in male sterility.

Animals↗
PubMed1991

Amblyospora sp. (Microspora, Amblyosporidae) infecting nerve ganglia of Culex pipiens (Diptera, Culicidae) from Egypt.

A species of Amblyospora-infecting neurones of Culex pipiens is described. Diplokaryotic meronts, which divided by binary fission, were distinguished at the electron microscope level by their unthickened plasma membranes. Sporonts with an electron-dense surface coat gave rise to eight uninucleate sporoblasts within a sporophorous vesicle, cytoplasmic division occurring at the quadrinucleate or octonucleate stages. Indications that nuclear fusion and chromosome reorganization occurred in merogony and sporogony were obtained by light microscopy but meiosis was not detected at the ultrastructural level. Spores were typical of Amblyospora, being ovoid when fresh, truncate when stained, and having an exospore of two membranous layers subtended by a thick amorphous layer, an electron-lucent endospore, an anisofilar polar filament, and a polaroplast comprised of an anterior region of close-packed lamellae and a posterior region of expanded sacs. The metabolic products in the sporophorous vesicle took the form of large globules, small globules with electron-dense borders, and fine granules. These were depleted in mature sporophorous vesicles, though a surface layer of fine granules on the spores may have been derived from them. Many stages were degenerate and it is suggested that C. pipiens may be an accidental host in which the parasite could develop suboptimally in nervous tissue only. Infections in larvae hatched from eggs in the laboratory indicate that vertical transmission occurs.

Animals↗