Ultrastructural localization of ATPase activity in cotton fiber during elongation

Summary The ultrastructural distribution of potassium chloride stimulated adenosine triphosphatase activity was investigated in the outer integument of a linted cultivar of cotton and a lintless (naked seed) mutant from one day preanthesis to eight days postanthesis by using a heavy metal simultaneous capture reaction technique. No enzyme activity other than in mitochondria was observed in the lintless mutant. In the linted cultivar no ATP-specific enzyme activity was seen in non-elongating epidermal cells, subepidermal cells of the outer integuments or any controls. As fiber initials started elongating, enzyme activity gradually appeared on the tonoplasts of enlarging vacuoles. Heavier lead phosphate deposits were observed on the membrane of small vacuoles compared to the tonoplast. This activity continued at least to eight days postanthesis. The enzyme inhibitor, N,N-dicyclohexylcarbodiimide inhibited, while KCl stimulated, tonoplast ATPase activity. The gradual increase of ATPase activity on the tonoplast of expanding fibers, but not on the tonoplasts of non-fiber cells, suggests the active transport of osmotically active compounds, presumably potassium and malate, into the vacuoles of expanding fibers. Fusion of smaller vacuoles with the large central vacuole indicates that these structures contribute additional membrane components along with their enzyme activity to the tonoplast of expanding fibers. The occurrence of ATPase activity, of ER-derived vesicular structures, and the organized pattern of deposition of these structures on the tonoplast indicate ER-originated ATPase activity. This study supports the theory of osmoregulation in cotton fiber where ATPase provides the energy for active accumulation of osmotically active compounds, (K⁺, malate) into the vacuoles, thereby generating and maintaining the turgor pressure required for fiber expansion.Abbreviations ATPase Adenosine triphosphatase - DCCD N,N-Di-cyclohexylcarbodiimide - EM Electron microscope - ER Endoplasmic reticulum - GP -Glycerophosphate - LC Lead citrate - PEP-Case Phosphoenolpyruvate carboxylase - UA Uranyl acetate     

The PtdIns 3-Kinase/Akt Pathway Regulates Macrophage-Mediated ADCC against B Cell Lymphoma

Macrophages are important effectors in the clearance of antibody-coated tumor cells. However, the signaling pathways that regulate macrophage-induced ADCC are poorly defined. To understand the regulation of macrophage-mediated ADCC, we used human B cell lymphoma coated with Rituximab as the tumor target and murine macrophages primed with IFNγ as the effectors. Our data demonstrate that the PtdIns 3-kinase/Akt pathway is activated during macrophage-induced ADCC and that the inhibition of PtdIns 3-kinase results in the inhibition of macrophage-mediated cytotoxicity. Interestingly, downstream of PtdIns 3-kinase, expression of constitutively active Akt (Myr-Akt) in macrophages significantly enhanced their ability to mediate ADCC. Further analysis revealed that in this model, macrophage-mediated ADCC is dependent upon the release of nitric oxide (NO). However, the PtdIns 3-kinase/Akt pathway does not appear to regulate NO production. An examination of the role of the PtdIns 3-kinase/Akt pathway in regulating conjugate formation indicated that macrophages treated with an inhibitor of PtdIns 3-kinase fail to polarize the cytoskeleton at the synapse and show a significant reduction in the number of conjugates formed with tumor targets. Further, inhibition of PtdIns 3-kinase also reduced macrophage spreading on Rituximab-coated surfaces. On the other hand, Myr-Akt expressing macrophages displayed a significantly greater ability to form conjugates with tumor cells. Taken together, these findings illustrate that the PtdIns 3-kinase/Akt pathway plays a critical role in macrophage ADCC through its influence on conjugate formation between macrophages and antibody-coated tumor cells.     

Co-transformation of Metarhizium anisopliae by electroporation or using the gene gun to produce stable GUS transformants

Abstract The potential of β-glucuronidase as a molecular marker for studying the environmental microbiology of entomopathogenic fungi was assessed. Metarhizium anisopliae was stably co-transformed with plasmids (pNOM102 and pBENA3) containing the β-glucuronidase and benomyl resistance (β-tubulin) genes, using both electroporation and biolistic delivery systems, and it was confirmed that the expressed phenotypes were not exhibited by ten randomly chosen indigenous North-American isolates. In spite of random and multiple integrations, the co-transformants showed normal growth rates and retained their pathogenicity to insects. β-Glucuronidase activity in the co-transformants was used to detect histochemically the presence of fungal hyphae in infected host insects ( Bombyx mori ) and thus provides a practical means of marking genetically engineered pathogens for field trials.     

Intercellular matrix in colonies of Candida.

The histochemistry and fine structure of typical colonies of six species of Candida were studied, using a total of 31 clinical isolates. The colonies consisted of viable and degenerate cells which lay in an intercellular matrix. This matrix was made up of amorphous, granular, and fibrillar components, the relative proportions and total amount of which varied from species to species. The cells of all species were surrounded by a zone of homogeneous amorphus material, which may be a highly cross-linked carbohydrate. This separated intact cells from irregularly distributed granular debris derived from the cytoplasm of degenerate cells. Focal cellular degeneration and associated granular debris were present within the colonies of all species and were most common in the surface layers of cells of colonies of C. albicans and C. tropicalis. The large amounts of intercellular matrix in this region formed a surface coat on colonies of these two species. Intercellular strands of cell wall material, and to a lesser extent other membranous elements from degenerate cells, formed a prominent fibrillar meshwork in the colonies of C. albicans and C. tropicalis, but were less common in those of C. pseudotropicalis and C. guilliermondii and seldom seen in those of C. parapsilosis and C. krusei.     

Immunologically reactive multiple species of mitochondrial coupling factor B

Earlier work had indicated that mitochondrial coupling factor B (FB) could be obtained with differing molecular weights, a highly active 13,000 form, a 29,200 form with low activity, and a partially purified 46,000 form with activity higher than the 29,200 form. We have analyzed FB preparations of different purity and after different types of treatment on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), followed by silver staining or immunostaining either with rabbit anti-FB serum or monoclonal FB antibody. Highly purified preparations which appear as single bands in SDS-PAGE develop additional higher molecular weight bands (silver staining), including a 48,000 and a 68,000 band, after lyophilization or repeated freezing and thawing or if subjected to SDS-PAGE in the absence of thiol compounds. FB prepared without addition of dithiothreitol and glycerol for stabilization also shows high molecular weight forms, although the active fractions are obtained consistently in the final gel filtration step of purification at a position corresponding to Mr = 13,000. When FB preparations are analyzed by immunoblots of SDS-PAGE using a monoclonal antibody to FB, fresh preparations of purified FB show a single band, while multiple bands are seen in samples which have been frozen and thawed repeatedly. Preparations made in the absence of dithiothreitol and glycerol also show cross-reactive forms of high molecular weight. Similar immunoblots using rabbit antiserum with mitochondria, its extracts, and partially purified FB preparations, all show the presence of several higher molecular weight forms. It is concluded that FB is probably a monomer in mitochondria, and it appears to undergo oligomerization after extraction and during purification.     

Serum progesterone and norethisterone levels following injection of norethisterone enanthate in different sites and doses

Serum progesterone (P) and norethisterone (NET) levels following injection of norethisterone enanthate (NETEN) were studied in 11 regularly menstruating women. In 6 subjects 200 mg NETEN was administered to the gluteal (IG) or deltoid (ID) region sequentially. The serum P levels remained anovulatory (less than 4 ng/mL) up to 12 weeks. Serum NET levels were similar whether injection was given IG or ID (paired t - test, NS). In 5 subjects given a 150-mg dose, serum P levels remained anovulatory up to 11 weeks. Serum NET levels declined faster than with the 200-mg dose, but the difference was not significant (Student's t - test, NS). Wide inter-subject variations were seen in both groups. NETEN therefore can effectively be administered in gluteal or deltoid regions. A lower dose of 150 mg may have contraceptive potential on an 8-week schedule.     

In situ assay of intracellular enzymes of yeast (Kluyveromyces fragilis) by digitonin permeabilization of cell membrane

The yeast, Kluyveromyces fragilis was permeabilized to a number of low-molecular-weight substrates using digitonin. The activities of intracellular yeast enzymes, viz., alcohol dehydrogenase (ADH), beta-galactosidase, glucose-6-phosphate dehydrogenase, aspartase, and hexokinase were found to be much higher in the permeabilized cells than the untreated cells. The optimum conditions for permeabilization with reference to ADH were 0.1% digitonin at 37 degrees C for 15 min. The ADH activity in permeabilized cells was several-fold higher than that in cell free extracts prepared by either physical or chemical methods.