Cloning, Expression, and Affinity Purification of RecombinantShigella flexneriInvasion Plasmid Antigens IpaB and IpaC

Shigella flexneriand related enteropathogenic bacteria are important agents of bacillary dysentery, a potentially life-threatening illness for children in underdeveloped regions of the world. Onset of shigellosis stems fromS. flexneriinvasion of colonic epithelial cells, leading to localized cell death and inflammation. Invasion plasmid antigens (Ipa) B, C, and D are three secreted proteins encoded by the large virulence plasmid ofS. flexnerithat have been implicated as essential effectors of this cell invasion process. These proteins are expressed as part of theipaoperon and are among the major targets of the host immune response to shigellosis. Biochemical characterization of the Ipa invasins has been complicated by the fact they have not been purified in the quantities needed for detailedin vitroanalysis. Here we describe the first cloning, expression, and extensive purification of IpaB and IpaC fusion proteins fromEscherichia colifor use in dissecting of the protein biochemistry ofS. flexneripathogenesis. A variety of approaches were used to prepare significant quantities of these proteins in their soluble forms, including the use of different host cell lines, modification of bacterial growth conditions, and the use of alternative plasmid expression vectors. Now that these Ipa proteins are available in a highly pure form, it will be possible to initiate studies on their important biological and immunological properties as well as their recruitment into high-molecular-weight protein complexes. Together with IpaD (purified as part of a previous study), these purified proteins will be useful for: (a) exploring properties of the host immune response toS. flexneriinvasion, (b) elucidating the specific biochemical properties that lead to pathogen internalization, (c) analyzing the importance of specific Ipa protein complexes in host cell invasion, and (d) monitoring, or perhaps even augmenting, the efficacy of live oral vaccines in human trials.     

OPTIMIZATION OF AN IMMUNOFLUORESCENT ASSAY OF THE INTERNAL ENZYME RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE (RUBISCO) IN SINGLE PHYTOPLANKTON CELLS1

Immunochemical probes are widely used to identih different species and to quantify and understand the role that different antigens play within cells. We optimized a single-cell immunofluorescent assay for the carbon fixation enzyme ribulose-1,5-bisphosphate carboxylase (Rubisco) in order to quantify the enzyme by flow cytometry in phytoplankton cells. The criteria for optimization of the immunofluorescent assay for Rubisco in single cells included maximization of Rubisco immunogenicity, minimization of Rubisco diffusion out of the cells, minimization of cell breakage, and maximization of the cell labeling. Several fixatives (cross-linkers and denaturing) and permeabilizing agents were tested on 26 species of phytoplankton. The only fixative / permeabilizing agent that fulfilled the criteria established for the assay was 96% ethanol. Phytoplankton cells collected from the field needed further treatment with a strong oxidant to permeabilize ethanolfixed cells and thus allow the antibody probe to access the Rubisco antigen. This study should have a general applicability to the study of other soluble photosynthetic antigens in single phytoplankton cells.     

The transfer of pyrethroid resistance resulting from crosses between resistant German cockroaches and susceptible Asian cockroaches

Crosses were made between the Asian cockroach,Blattella asahinai Mizukubo, and resistant strains of the German cockroach,B. germanica (L.), to assess the transfer of pyrethroid resistance to the progeny and to study the inheritance mechanism(s) involved. It was shown that the strain of Asian cockroaches studied was susceptible to four pyrethroids. F₁ progeny were essentially susceptible to the same compounds. Tests with F₂ progeny and those from backcrosses to the resistant parent indicated that the data for each pyrethroid fit an hypothesis of simple, autosomal, nearly completely recessive inheritance. The results are discussed from the standpoint of the impact of the Asian genome on the inheritance mechanism(s).     

The mATPase histochemical profile of rat type IIX fibres: correlation with myosin heavy chain immunolabelling

Summary In the present study we report a novel histochemical method which, by sequential pre-incubations in alkaline and acidic media, selectively differentiates muscle fibres expressing myosin heavy chain IIX, on the basis of a specific profile for myofibrillar actomyosin ATPase (mATPase) activity. The enzyme reactions were tested for specificity by means of anti-myosin heavy chain monoclonal antibodies, which were characterized on Western blots of muscle homogenates. Enzyme histochemical reactions with the traditional pH buffers were compared to those of the new method and, in conjunction with the immunoreactions, used to confirm the relationship between MyHC expression and the distinct profiles for mATPase. Imrnunohistochemical reactions demonstrated that the new method only differentiates those fibres expressing myosin heavy chain IIX. The method revealed a continuum in which the intermediate staining intensities corresponded to hybrid fibres expressing myosin heavy chain IIX in combination with either the IIA or IIB forms. Quantitative histochemistry and immunohistochemistry (by image analysis), used to examine the relationship between staining intensities for mATPase and amounts of myosin heavy chain IIX expression, revealed that the new method discriminates well between hybrid fibres expressing variable amounts of the IIX isoform (r² = 0.93).     

DNA topology: Topoisomerases keep it simple

The ability of type II DNA topoisomerases to perturb the equilibrium distributions of DNA topoisomers is a consequence of their ability to hydrolyse ATP. A sliding mechanism of topoisomerase action has been proposed to account for this phenomenon.     

Neocentromere-mediated Chromosome Movement in Maize

Neocentromere activity is a classic example of nonkinetochore chromosome movement. In maize, neocentromeres are induced by a gene or genes on Abnormal chromosome 10 (Ab10) which causes heterochromatic knobs to move poleward at meiotic anaphase. Here we describe experiments that test how neocentromere activity affects the function of linked centromere/kinetochores (kinetochores) and whether neocentromeres and kinetochores are mobilized on the spindle by the same mechanism. Using a newly developed system for observing meiotic chromosome congression and segregation in living maize cells, we show that neocentromeres are active from prometaphase through anaphase. During mid-anaphase, normal chromosomes move on the spindle at an average rate of 0.79 μm/min. The presence of Ab10 does not affect the rate of normal chromosome movement but propels neocentromeres poleward at rates as high as 1.4 μm/min. Kinetochore-mediated chromosome movement is only marginally affected by the activity of a linked neocentromere. Combined in situ hybridization/immunocytochemistry is used to demonstrate that unlike kinetochores, neocentromeres associate laterally with microtubules and that neocentromere movement is correlated with knob size. These data suggest that microtubule depolymerization is not required for neocentromere motility. We argue that neocentromeres are mobilized on microtubules by the activity of minus end–directed motor proteins that interact either directly or indirectly with knob DNA sequences. C urrent models suggest that chromosomes move by a combination of forces generated by microtubule disassembly (Inoue and Salmon, 1995; Waters et al., 1996) and the activity of molecular motors (Vernos and Karsenti, 1996; Yen and Schaar, 1996). Microtubule disassembly generates a constant poleward force; while molecular motors can generate force in either poleward or away-from-pole directions, depending on the characteristics of the motor protein. Both plus and minus end–directed microtubule-based motors are localized to kinetochores (Hyman and Mitchison, 1991). Immunolocalization experiments indicate that mammalian kinetochores contain the minus end– directed motor dynein throughout metaphase and anaphase (Pfarr et al., 1990; Steuer et al., 1990). The kinesin-like proteins CENP-E, which has a transient kinetochore localization in animals, and MCAK, which is localized between the kinetochore plates of mammalian chromosomes, are also thought to generate and/or regulate chromosome movement (Yen et al., 1992; Lombillo et al., 1995; Wordeman and Mitchison, 1995).In addition to the molecular motors on kinetochores, several kinesin-like proteins are localized to chromosome arms (Vernos and Karsenti, 1996). Two subfamilies of arm-based motors have been identified in animals: the NOD subfamily (Afshar et al., 1995; Tokai et al., 1996) and the Xklp1/chromokinesin subfamily (Vernos et al., 1995; Wang and Adler, 1995). Both Nod and Xklp1 are required for positioning chromosomes on the metaphase plate, suggesting that they encode plus end–directed motors (Afshar et al., 1995; Vernos et al., 1995). Other evidence suggests that minus end–directed motors interact with chromosome arms. In the plant Haemanthus, a poleward force acts along chromosome arms during metaphase (Khodjakov et al., 1996), and forces propelling chromosome arms poleward have been detected during anaphase in crane fly spermatocytes (Adames and Forer, 1996). Little is known about how poleward arm motility at metaphase–anaphase affects the fidelity or rate of chromosome segregation.The neocentromeres of maize (Rhoades and Vilkomerson, 1942) provide a particularly striking example of poleward chromosome arm motility. In the presence of Abnormal chromosome 10 (Ab10),¹ heterochromatic DNA domains known as knobs are transformed into neocentromeres and mobilized on the spindle (Rhoades and Vilkomerson, 1942; Peacock et al., 1981; Dawe and Cande, 1996). Knobs are primarily composed of a tandem 180-bp repeat (Peacock et al., 1981) which shows homology to a maize B centromere clone (Alfenito and Birchler, 1993). A characteristic feature of neocentromeres is that they arrive at the spindle poles in advance of centromeres; in extreme cases the neocentromere-bearing chromosome arms stretch towards the poles (Rhoades and Vilkomerson, 1942; Rhoades, 1952). A recently identified mutation (smd1) demonstrates that a trans-acting factor(s) encoded on Ab10 is essential for converting the normally quiescent heterochromatic knobs into active neocentromeres (Dawe and Cande, 1996).Here we use neocentromeres as a model for understanding the mechanisms and importance of nonkinetochore chromosome movement. As a part of our analysis, we developed a four-dimensional system for observing chromosome segregation in living meiocytes. Our experiments were designed to determine (a) how poleward arm motility affects the rate and fidelity of chromosome segregation; and (b) whether the mechanism of neocentromere motility is comparable to the mechanism of kinetochore motility.