The Use of Nonaqueous Fractionation to Assess the Ionic Composition of the Apoplast during Fruit Ripening

We have examined the possibility that pectin solubilization and cell separation in fruit may be due to organic acids disrupting calcium bridges between pectic polysaccharides. With fruit from a wild tomato (Lycopersicon pimpinellifolium [Dunal]) we demonstrated the validity of a nonaqueous fractionation method to obtain reliable estimates of the ionic content of the apoplast. In unripe fruit no organic acids were associated with the cell wall, which contained 67% of the total calcium and 47% of the magnesium. In ripe fruit 4% of the malate, 10% of the citrate, and 15% of the oxalate were estimated to be in the cell wall, together with 84% of the calcium and 52% of the magnesium. In contrast to the cultivated tomato, we did not find a consistent decrease in the degree of methyl esterification between unripe and ripe fruit, and an overall average of 75% was observed. In the cell walls of ripe fruit the ratio of calcium:magnesium:organic acid:unesterified uronic acid, on the basis of charge, was 15:4:4:16. The use of a computer program to predict the proportions of different ionic species in complex mixtures suggested that in ripe fruit 70% of the unesterified uronic acid would be complexed with calcium. Our results show that organic acids do not accumulate in the cell wall sufficiently to disrupt calcium cross-linking, nor is the calcium removed from the wall into the cell. We therefore conclude that organic acids do not contribute to cell separation during the ripening of tomato fruit.     

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).     

Partial restoration of impaired alpha1-adrenergic responsiveness in parotid cells of aged rats by S-adenosylmethionine treatment

The age related decrease in alpha₁-adrenergic stimulated inositol 1,4,5 trisphosphate (IP₃) production in parotid cells of aged rats can be partially restored by treatment with S-adenosylmethionine (SAM). This effect is completely blocked by S-adenosyl homocysteine (SAH) and occurs in association with an increase in the conversion of phosphatidylethanolamine to phosphatidylcholine and a decrease in membrane viscosity. In contrast, SAM treatment actually inhibits stimulated IP₃ production in cells of young rats. The membrane viscosity of these cells is lower than that of those from aged rats. Although conversion of phosphatidylethanolamine to phosphatidylcholine is enhanced, no further decrease in membrane viscosity is elicited in young cell preparations. These findings suggest that age changes in the membrane environment may result in impaired alpha₁-adrenergic signal transduction and that such alterations may be at least partially reversible by SAM treatment.     

Trace metal bioavailability in municipal solid waste and sewage sludge composts

A sequential extraction procedure was employed to determine the soil fractions, and assess plant availability of Cr, Cu, Ni, Pb and Zn in a Glynwood silt loam amended with five rates (0, 30, 60, 120 and 240 metric tons/ha) of composted municipal solid waste (CMSW) or composted sewage sludge (CSS) cropped to oats (Avena sativa). The application of the composts tended to shift the solid phase forms of the metals away from those extractable with HNO₃ to those extractable with NaOH and EDTA. The more labile fractions (KNO₃ and H₂O extracts) of the metals typically decreased with application of CMSW and CSS. Crop dry matter increased at the 30 and 60 MT/ha CMSW rates by 142 and 152%, respectively, after which yields declined to below control values. Yields at all rates of CSS declined. The CMSW and CSS had an insignificant effect on concentrations of Cr and Pb in oat tissue, but tissue levels of Cu, Ni and Zn increased with increased rate of compost application.     

Polyamine derivatives as inhibitors of trypanothione reductase and assessment of their trypanocidal activities

Trypanothione reductase (TR) occurs exclusively in trypanosomes and leishmania, which are the etiological agents of many diseases. TR plays a vital role in the antioxidant defenses of these parasites and inhibitors of TR have potential as antitrypanosomal agents. We describe the syntheses of several spermine and spermidine derivatives and the inhibiting effects of these compounds on T. cruzi TR. All of the inhibiting compounds displayed competitive inhibition of TR-mediated reduction of trypanothione disulfide. The three most effective compounds studied were N⁴,N⁸-bis(3-phenylpropyl)spermine (12), N⁴,N⁸-bis(2-naphthylmethyl)spermine (14), and N¹,N⁸-bis(2-naphthylmethyl)spermidine (21), with K_i values of 3.5, 5.5 and 9.5 μM, respectively. Compounds 12, 14, and 21 were found to be potent trypanocides in vitro with IC₅₀ values ranging from 0.19 to 0.83 μM against four T. brucei ssp. strains. However, these compounds did not prolong the lives of mice infected with trypanosomes. This work indicates that certain polyamine derivatives which target a unique pathway in Trypanosomatidae have potential as antitrypanosomal agents.     

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.     

Production of gliotoxin during the pathogenic state in turkey poults byAspergillus fumigatus Fresenius

Turkey poults were given either of two different dosages of two different gliotoxin-producing strains ofAspergillus fumigatus. Infected lung tissue was examined postmortem for the presence of gliotoxin. Gliotoxin was found in lung tissue of ten poults infected with one strain and in seven of ten poults infected with the other strain. Concentrations of gliotoxin in the tissue exceeded 6 ppm in some of the infected tissues. The concentration of gliotoxin found in infected tissue did not appear to be correlated with the dosage of organism given. Considering the pathologic changes observed in turkey poults with aspergillosis and the production of gliotoxin during the pathogenic state in turkey poults, gliotoxin is considered likely to be involved in avian aspergillosis. Disclaimer: Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Mannosyltransferase activities in membranes from various yeast strains

In the yeast Golgi compartments, at least five, and potentiallyseveral additional mannosyltransferases are involved in elongatingto ‘mannan’ the core Man₈GlcNAc₂ oligosac-charidetrimmed from GlC₃Man₉GlcNAc₂ in the endoplasmic reticulum. Structuralstudies on oligosaccharides from alg3 mutant yeast, which lackthe four upper arm mannoses donated by Man-P-Dol (where Dolis dolichol), verified that the new