Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in aphids of the genus Sitobion (Hemiptera: Aphididae)

Polymerase chain reaction (PCR) products corresponding to 803 bp of thecytochrome oxidase subunits I and II region of mitochondrial DNA (mtDNACOI-II) were deduced to consist of multiple haplotypes in three Sitobionspecies. We investigated the molecular basis of these observations. PCRproducts were cloned, and six clones from one individual per species weresequenced. In each individual, one sequence was found commonly, but alsotwo or three divergent sequences were seen. The divergent sequences wereshown to be nonmitochondrial by sequencing from purified mtDNA and Southernblotting experiments. All seven nonmitochondrial clones sequenced tocompletion were unique. Nonmitochondrial sequences have a high proportionof unique sites, and very few characters are shared betweennonmitochondrial clones to the exclusion of mtDNA. From these data, weinfer that fragments of mtDNA have been transposed separately (probablyinto aphid chromosomes), at a frequency only known to be equalled inhumans. The transposition phenomenon appears to occur infrequently or notat all in closely related genera and other aphids investigated. Patterns ofnucleotide substitution in mtDNA inferred over a parsimony tree are verydifferent from those in transposed sequences. Compared with mtDNA,nonmitochondrial sequences have less codon position bias, more evenexchanges between A, G, C and T, and a higher proportion of nonsynonymousreplacements. Although these data are consistent with the transposedsequences being under less constraint than mtDNA, changes in thenonmitochondrial sequences are not random: there remains significantposition bias, and probable excesses of synonymous replacements and ofconservative inferred amino acid replacements. We conclude that aproportion of the inferred change in the nonmitochondrial sequencesoccurred before transposition. We believe that Sitobion aphids (and otherspecies exhibiting mtDNA transposition) may be important for studying themolecular evolution of mtDNA and pseudogenes. However, our data highlightthe need to establish the true evolutionary relationships between sequencesin comparative investigations.     

Effect of dilution rate on the release of pertussis toxin and lipopolysaccharide ofBordetella pertussis

Summary The kinetics ofBordetella pertussis growth was studied in a glutamate-limited continuous culture. Growth kinetics corresponded to Monod's model. The saturation constant and maximum specific growth rate were estimated as well as the energetic parameters, theoretical yield of cells and maintenance coefficient. Release of pertussis toxin (PT) and lipopolysaccharide (LPS) were growth-associated. In addition, they showed a linear relationship between them. Growth rate affected neither outer membrane proteins nor the cell-bound LPS pattern.Nomenclature X cell concentration (g L^–1) - specific growth rate (h^–1) - _m maximum specific growth rate (h^–1) - D dilution rate (h^–1) - S concentration of growth rate-limiting nutrient (glutamate) (mmol L^–1 or g L^–1) - Ks substrate saturation constant (mol L^–1) - m_s maintenance coefficient (g g^–1 h^–1) - Y_x/s theoretical yield of cells from glutamate (g g^–1) - Y_x/s yield of cells from glutamate (g g^–1) - Y_PT/s yield of soluble PT from glutamate (mg g^–1) - Y_KDO/s yield of cell-free KDO from glutamate (g g^–1) - Y_PT/x specific yield of soluble PT (mg g^–1) - Y_KDO/x specific yield of cell-free KDO (g g^–1) - q_PT specific soluble PT production rate (mg g^–1 h^–1) - q_KDO specific cell-free KDO production rate (g g^–1 h^–1)     

Fc receptor-mediated immunity against Bordetella pertussis.

The relevance of specific Abs for the induction of cellular effector functions against Bordetella pertussis was studied. IgG-opsonized B. pertussis was efficiently phagocytosed by human polymorphonuclear leukocytes (PMN). This process was mediated by the PMN IgG receptors, FcgammaRIIa (CD32) and FcgammaRIIIb (CD16), working synergistically. Furthermore, these FcgammaR triggered efficient PMN respiratory burst activity and mediated transfer of B. pertussis to lysosomal compartments, ultimately resulting in reduced bacterial viability. Bacteria opsonized with IgA triggered similar PMN activation via FcalphaR (CD89). Simultaneous engagement of FcalphaRI and FcgammaR by B. pertussis resulted in increased phagocytosis rates, compared with responses induced by either isotype alone. These data provide new insights into host immune mechanisms against B. pertussis and document a crucial role for Ig-FcR interactions in immunity to this human pathogen.     

Initial verification of the resistance management strategy for Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) in Australia

Shortly after the initial detection of western flower thrips (WFT), Frankiniella occidentalis (Pergande), in Australia during 1993 a resistance management strategy based on the alternation of chemical groups was implemented. This study aimed to verify this strategy by field testing α-cypermethrin against WFT with and without chemical alternation. Up to 114 times α-cypermethrin resistance (at LC50) was detected and resistance increased with and without chemical alternation; however, chemical alternation did significantly reduce numbers of thrips compared with a nonalternation strategy. Resistance has the potential to undermine the sustainable use of those chemicals to which there is no current detectable resistance. Consequently, chemicals with a high frequency and level of resistance against WFT need to be identified through monitoring and quickly eliminated from WFT chemical control recommendations.     

Quantitation of Adenylate Cyclase ofBordetella pertussisby Enzyme Linked Immunosorbent Assay

Bordetella pertussisproduces extracytoplasmic adenylate cyclase toxin (AC) which has received considerable attention as a potential vaccine candidate. Great interest from laboratories involved in production, purification and quality control of acellular pertussis vaccine is focused on finding an appropriate technique for rapid and accurate quantitation of AC antigen. In this paper a competitive ELISA is proposed. A polystyrene microplate coated with purified AC was incubated with the sample to be tested plus anti-AC serum. The bound anti-AC antibodies were measured by sequential reaction with alkaline phosphatase-labelled anti-mouse IgG and p-nitrophenylphosphate. This method showed high specificity, with the 50% inhibition corresponding to 4 μg/ml of AC. It also proved to be useful to assess the presence of AC in culture supernatants, with high reproducibility.     

A simple method for studying whole sections of rice grain

We developed a new and simple method to collect sections of a whole brown rice kernel for investigation of histological properties. A single kernel of rice was dehydrated through a graded ethanol series, transferred to xylene, and embedded in paraffin. During sectioning of the blocks using a rotary microtome, we used a special adhesive tape to collect and place the sections on slides so they remained flat. The use of the adhesive tape technique combined with autofluorescence characteristics allowed us to visualize cell walls throughout an entire longitudinal or transverse section of a whole rice kernel. We obtained scanning electron microscopy images of the sections to determine section quality.     

Environment and virulence factors of Vibrio cholerae strains isolated in Argentina

AIMS: To determine the presence of Vibrio cholerae in different areas of Argentina in three sample types, to determine the composition of planktonic communities in areas at which this pathogen was detected and to characterize the virulence properties and antimicrobial resistance of the recovered environmental isolates. METHODS AND RESULTS: Water and plankton samples were collected in marine, brackish and freshwater environments. Vibrio cholerae non-O1, non-O139 was isolated in 36.1% of the samples analysed. The micro-organism was detected in freshwater but not in marine or brackish samples. No relationship was found between isolation of V. cholerae and presence of any species of plankton. All the isolates presented very similar virulence profiles by PCR, lacking ctxA and tcpA El Tor and containing hlyA (98.7%), rtxA (99.0%), toxR (98.7%) and stn-sto (1.9%). Resistance to ampicillin was found in both Tucumán (21%) and Buenos Aires isolates (45%). CONCLUSIONS: We identified two geographic areas in Argentina where V. cholerae was present: freshwaters of the rivers from Tucumán and the Río de la Plata. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of V. cholerae strains in the environment, carrying both virulence factors and resistance to antimicrobial agents, highlight the need for a continuous and active surveillance of this pathogen.     

Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification

The present study investigated the effect of estrous cow serum (ECS) during culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. Embryos were derived from in vitro maturation (IVM) and in vitro fertilization (IVF) of abbatoir-derived oocytes. At Day 3, embryos were cultured in three different media: Charles Ronsenkrans medium + amino acids (CR1aa; without bovine serum albumin (BSA)) + 5% estrous cow serum (CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA) or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). At 7.5 d post-insemination (PI), blastocyst yield and quality were evaluated; blastocysts and expanded blastocysts from each media were cryopreserved by Open Pulled Straw (OPS) vitrification method or slow freezing (1.5 M ethylene glycol, EM). Total blastocyst yield did not differ among CR1-ECS, CR1-BSA and CR1-ECS-BSA (30.9, 33.1 and 32.9%, respectively, P < 0.05). Embryo survival (hatching rate) was higher in vitrified versus slow-frozen embryos (43% versus 12%, respectively, P < 0.01), and in embryos cultured in CR1-BSA (40.3%) compared with those cultured in serum-containing media (CR1-ECS, 21.5% and CR1-ECS-BSA, 19.8%; P < 0.01). In conclusion: (a) it was possible to produce in vitro bovine embryos in serum-free culture medium without affecting blastocyst yield and quality; (b) serum-free medium produced the best quality embryos (in terms of post-cryopreservation survival); and (c) vitrification yielded the highest post-cryopreservation survival rates, regardless of the presence of serum in the culture medium.