Growth of Phytomonas serpens in a Defined Medium; Nutritional Requirements

ABSTRACT. Three strains of Phytomonas serpens two from tomatoes, Lycopersicon esculentum one from the insect Phtia picta (Hemiptera, Coreidae), were cultivated in a chemically defined medium developed from a defined medium for cultivating insect flagellates. Besides organic growth factors required by other insect trypanosomatids this flagellate requires, serine and inositol. Glutamine stimulates growth, and, surprisingly, does not require heme.     

The tympanal hearing organ of the parasitoid fly Ormia ochracea (Diptera, Tachinidae, Ormiini)

Tympanate hearing has evolved in at least 6 different orders of insects, but had not been reported until recently in the Diptera. This study presents a newly discovered tympanal hearing organ, in the parasitoid tachinid fly, Ormia ochracea. The hearing organ is described in terms of external and internal morphology, cellular organization of the sensory organ and preliminary neuroanatomy of the primary auditory afferents. The ear is located on the frontal face of the prothorax, directly behind the head capsule. Conspicuously visible are a pair of thin cuticular membranes specialized for audition, the prosternal tympanal membranes. Directly attached to these membranes, within the enlarged prosternal chamber, are a pair of auditory sensory organs, the bulbae acusticae. These sensory organs are unique among all auditory organs known so far because both are contained within an unpartitioned acoustic chamber. The prosternal chamber is connected to the outside by a pair of tracheae. The cellular anatomy of the fly's scolopophorous organ was investigated by light and electron microscopy. The bulba acustica is a typical chordotonal organ and it contains approximately 70 receptor cells. It is similar to other insect sensory organs associated with tympanal ears. The similarity of the cellular organization and tympanal morphology of the ormiine ear to the ears of other tympanate insects suggests that there are potent constraints in the design features of tympanal hearing organs, which must function to detect high frequency auditory signals over long distances. Each sensory organ is innervated by a branch of the frontal nerve of the fused thoracic ganglia. The primary auditory afferents project to each of the pro-, meso-, and metathoracic neuropils. The fly's hearing organ is sexually dimorphic, whereby the tympanal membranes are larger in females and the spiracles larger in males. The dimorphism presumably reflects differences in the acoustic behavior in the two sexes.     

Comparison of Vero cell assay and PCR as indicators of the presence of verocytotoxigenic Escherichia coli in bovine and human fecal samples.

Comparisons were made between Vero cell assay (VCA) and PCR as indicators for the detection of verocytotoxigenic Escherichia coli (VTEC; also known as Shiga-like toxin-producing E. coli) and as predictors of VTEC isolation from bovine and human fecal samples. Fecal samples were collected as part of a survey on the prevalence of VTEC on dairy farms in southern Ontario (J. B. Wilson et al., J. Infect. Dis., 174:1021-1027, 1996). A total of 2,655 samples were examined by VCA and PCR, 2,153 originating from cattle and 502 originating from humans. Overall, 36.2% of the samples were positive in the VCA and 38.7% were positive by PCR. Of the VCA-positive samples screened, 41.6% yielded a VTEC isolate. For both human and bovine samples, a significant positive association between PCR result and VCA titer (P = 0.0001) was found. In addition, there was a significant positive association between the PCR result and VTEC isolation from VCA-positive samples for cattle (odds ratio = 9.1, P < 0.0001). For bovine samples positive in the VCA, VCA titer was significantly associated with the probability of obtaining a VTEC isolate. Agreement between VCA and PCR was good for both bovine and human samples (kappa = 0.69 and 0.64, respectively). The sensitivity and specificity of the PCR with respect to the VCA for bovine samples were 82.0 and 86.5%, respectively, and those for human samples were 59.3 and 98.1%, respectively. Although correlation between VCA and PCR results was not absolute, when used in conjunction, these tests complemented one another as predictors of VTEC isolation.     

Membrane Skeletal Proteins and Their Integral Membrane Protein Anchors are Targets for Tyrosine and Threonine Kinases in Euglena

ABSTRACT. Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [³²P]-orthophosphate or γ-[³²P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.     

Assembly of mitochondrial ribonucleoprotein complexes involves specific guide RNA (gRNA)-binding proteins and gRNA domains but does not require preedited mRNA.

RNA editing in kinetoplastids probably employs a macromolecular complex, the editosome, that is likely to include the guide RNAs (gRNAs) which specify the edited sequence. Specific ribonucleoprotein (RNP) complexes which form in vitro with gRNAs (H. U. Göringer, D. J. Koslowsky, T. H. Morales, and K. D. Stuart, Proc. Natl. Acad. Sci. USA, in press) are potential editosomes or their precursors. We find that several factors are important for in vitro formation of these RNP complexes and identify specific gRNA-binding proteins present in the complexes. Preedited mRNA promotes the in vitro formation of the four major gRNA-containing RNP complexes under some conditions but is required for the formation of only a subcomponent of one complex. The 5' gRNA sequence encompassing the RYAYA and anchor regions and the 3' gRNA oligo(U) tail are both important in complex formation, since their deletion results in a dramatic decrease of some complexes and the absence of others. UV cross-linking experiments identify several proteins which are in contact with gRNA and preedited mRNA in mitochondrial extracts. Proteins of 25 and 90 kDa are highly specific for gRNAs, and the 90-kDa protein binds specifically to gRNA oligo(U) tails. The gRNA-binding proteins exhibit a differential distribution between the four in vitro-formed complexes. These experiments reveal several proteins potentially involved in RNA editing and indicate that multiple recognition elements in gRNAs are used for complex formation.     

Locus Heterogeneity for Waardenburg Syndrome is Predictive of Clinical Subtypes

Waardenburg syndrome (WS) is a dominantly inherited and clinically variable syndrome of deafness, pigmentary changes, and distinctive facial features. Clinically, WS type I (WS1) is differentiated from WS type II (WS2) by the high frequency of dystopia canthorum in the family. In some families, WS is caused by mutations in the PAX3 gene on chromosome 2q. We have typed microsatellite markers within and flanking PAX3 in 41 WS1 kindreds and 26 WS2 kindreds in order to estimate the proportion of families with probable mutations in PAX3 and to study the relationship between phenotypic and genotypic heterogeneity. Evaluation of heterogeneity in location scores obtained by multilocus analysis indicated that WS is linked to PAX3 in 60% of all WS families and in 100% of WS1 families. None of the WS2 families were linked. In those families in which equivocal lod scores (between −2 and +1) were found, PAX3 mutations have been identified in 5 of the 15 WS1 families but in none of the 4 WS2 families. Although preliminary studies do not suggest any association between the phenotype and the molecular pathology in 20 families with known PAX3 mutations and in four patients with chromosomal abnormalities in the vicinity of PAX3, the presence of dystopia in multiple family members is a reliable indicator for identifying families likely to have a defect in PAX3.     

Refining the genetic map for the region flanking the X-linked hypophosphataemic rickets locus (Xp22.1–22.2)

An analysis of five of the most common cystic fibrosis (CF) mutations worldwide (F-508, R-553X, G-551D, N-1303K and G-542X) was performed in 36 Chilean patients. Polymerase chain reaction (PCR) amplification of the DNA followed by allele specific restriction enzyme analysis was used for detection. The overall frequencies of the mutations in the chromosomes analyzed were 29.2% for F-508 and 4.2% for R-553X (n=72). The G-542X, G-551D and N-1303 K mutations were absent in the Chilean sample. Our data suggest however that F-508 is not the most common CF mutation in Chilean patients. F-508 and R-553X account for only 33.4% of the alleles; 66.6% of them do not respond to the probes used and still remain uncharacterized.     

Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi

Borrelia burgdorferi is a spirochete pathogen transmitted among warm- blooded hosts by ixodid ticks. Frequency-dependent selection for variant outer-surface proteins might be expected to arise in this species, since rare variants are more likely to avoid immune surveillance in previously infected hosts. We sequenced the OspA and OspB genes of nine North American strains and compared them with nine strains previously described. For each gene, the mean number of synonymous substitutions per synonymous site and the mean number of nonsynonymous substitutions per nonsynonymous site show only a twofold excess of silent mutations. Synonymous rates vary widely along the OspB protein. Some regions show a significant excess of silent substitutions, while divergence in other regions is constrained by biased base composition or selection. The presence, in antigenically important regions of the protein, of significant variation among strains, as well as evidence for recombination among strains, should be considered in attempts to develop vaccines against this disease.      

In vitro culture of vanhoutte's spirea explants from ‘secondary cultures’ and dormant stems forced in solutions containing plant growth regulators

Explants from new growth of forced dormant stems and secondary cultures of Vanhoutte's spirea were cultured on Linsmaier and Skoog (L.S.) medium containing benzyladenine (BA), indoleacetic acid (IAA), thidiazuron (TDZ), and zeatin. The dormant stems were forced by immersing their basal portions in forcing solutions containing 626 µM 8-hydroxyquinoline citrate (8-HQC) and 2% sucrose. BA and gibberellic acid (GA₃) were also added into the forcing solutions to determine if explants obtained from the new growth will benefit from this treatment when culturedin vitro.L.S. medium supplemented with 5 µM BA alone, 5 µM BA plus 1 or 5 µM IAA, and 0.5 or 0.75 µM TDZ alone produced the best shoot proliferation for both sources of explants. BA and GA₃ appeared to be taken up from the forcing solution by the new softwood growth. BA in the forcing solution stimulatedin vitro shoot proliferation in different degrees depending on the period of treatment, while GA₃ caused lessin vitro shoot proliferation. It is proposed that forcing solutions containing plant growth regulators (P.G.R.) are a useful approach for manipulating responses of plant tissues culturedin vitro.     

Characterisation of a 34 kD protein from potato cyst nematodes, using monoclonal antibodies with potential for species diagnosis

Two monoclonal antibodies, which differentially recognise the two species of potato cyst nematodes (PCN), Globodera pallida and G. rostochiensis, are described. They have been shown to have potential for quantification of these two species, recognising proteins of the same molecular weight (34 kD) in both species. Further investigation showed these proteins to have isoelectric points at pH values of 5.7 in G. pallida and 5.9 in G. rostochiensis, in common with the proteins used by Fleming & Marks (1983) to differentiate the species of PCN. They are likely to be structurally very similar, with the same physiological function (and therefore similar concentrations) in the two species. In cross-reactivity tests with a wide range of soil nematode species, the antibodies reacted strongly only with species of the genus Globodera, and thereby confirmed their potential as the basis of a quantitative immunoassay likely to be useful in management of PCN populations.