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31.
Summary Heat of dilution ofHalobacterium halobium was measured when thick pastes of the bacteria, harvested throughout a complete growth cycle, were lysed by mixing with 40 times their volume of water in a microcalorimeter. A series of comparative measurements was made with pastes of bacteria previously disrupted by freezing and thawing but otherwise identical to the pastes of whole bacteria. The frozen-thawed pastes gave endothermic values some 18% greater than those obtained with intact bacteria; the difference was highly significant. Evidence was obtained that the mechanical component of bursting did not contribute to the difference between whole and lysed bacteria. On the other hand, when a correction was applied for heat of mixing of intracellular salts with extracellular NaCl, such as occurs when the bacteria lyse, the difference between whole and disrupted organisms was largely eliminated from exponential phase halobacteria but not from those harvested in stationary phase. It is concluded that there is no evidence, as reflected in heat of dilution, of abnormal solution properties of the cytosol of young halobacteria, which are rich in potassium. On the other hand, and paradoxically, some doubt remains about stationary phase organisms whose cytosol has a much higher Na+ content (and Na/K+ ratio) than the cytosol of exponential phase bacteria. 相似文献
32.
Kiem-Phong Vo Mark J. Miller E.Peter Geiduschek Chris Nielsen Arthur Olson 《Analytical biochemistry》1981,112(2):258-271
New methods and computer programs are described which enable one to analyze autoradiograms produced by two-dimensional gel electrophoresis. These programs are completely automatic with respect to finding spots resolved by such gels and quantitating the radioactivity in them. Semiautomatic programs have also been developed to match the spot patterns of different autoradiograms, and to follow the synthesis of any individual polypeptide through a series of gels. 相似文献
33.
Summary New cytochemical method, based on biochemical experiments, was elaborated for the ultrastructural localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31). The procedure was used to study the saprophytic submerged mycelium of the ascomycetous fungusClaviceps purpurea Tul. producing clavine alkaloids. The pelleted mycelium was fixed in ice cold 3% glutaraldehyde in 50 mM cacodylate buffer pH 7.2 and washed repeatedly in the same cold buffer. The reaction mixture contained 100 mM Tris-HCl buffer pH 9.0, 10 mM phospho(enol)pyruvate, 30 mM sodium potassium tartrate, 3 mM Pb(NO3)2, 60 mM MgCl2 and 30 mM NaHCO3. Enzyme activity was localized in vacuoles, particularly inside lipid globules (spherosomes) and less frequently in membranous vesicles. Acetyl-CoA activated PEP-carboxylase both in cell free extracts and in the cytochemical staining. Aspartate inhibited the enzyme in the biochemical assay with coupled malate dehydrogenase system; the cytochemical reaction was not influenced, probably due to the interference of asparagine synthase (EC 6.3.1.1). 相似文献
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Summary The ultrastructural localization of acetyl-CoA carboxylase activity was studied in two strains of the ascomycetous fungus Claviceps purpurea differing in the ergot alkaloid synthesis. Mycelia were harvested by centrifugation of saprophytic submerged cultures, fixed in cold 3% glutaraldehyde in 0.05 M cacodylate buffer pH 7.2 and washed repeatedly in the same buffer. The incubation medium of Yates et al. (1969) had to be modified in the molarity of ATP. The best results were obtained with a medium of the following composition: 50 mM cacodylate buffer pH 7.2, 4 mM ATP, 3.5 mM lead nitrate, 13.5 mM sodium citrate, 3.75 mM sodium bicarbonate, 1.25 mM manganese chloride, 0.4 mM acetyl-CoA and 2 mM biotin. The fixation is a prerequisite for a distinct localization. The enzyme activity was detected only in cells producing high amount of clavine alkaloids. It was confined to the membranes of endoplasmic reticulum and their derivatives: tonoplast of vacuoles, tiny vesicles and amorphous material inside vacuoles. The reaction product was very fine and localized in both leaflets of the membranes. The specificity of the reaction was confirmed by negative results in control preparations: boiled cells incubated in the complete medium, cells incubated in the medium supplemented with avidin or in the media from which either ATP, or acetyl CoA, or sodium bicarbonate, or biotin were omitted. It is suggested that the activity of acetyl-CoA carboxylase is linked to the synthesis of clavine alkaloid precursors which occurs in the endoplasmic reticulum and its derivatives.With technical assistance of J. Martínková 相似文献
36.
Ultrathin sectioning of submerged mycelium of Claviceps purpurea Tul. producing clavine alkaloids revealed yeast-like budding resulting in asexual sporesblastospores. These deciduous spores were born by extended hyphal cells and retained the same ultrastructure of cell organelles. Both the extended hyphae and the blastospores resembled the cells of ergot sclerotial tissue. A surface culture of C. purpurea Tul. producing no alkaloids was used as a reference. 相似文献
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Huan Chen Migara Kavishka Jayasinghe Eric Yew Meng Yeo Zhiyuan Wu Marco Pirisinu Waqas Muhammad Usman Thach Tuan Pham Kah Wai Lim Nhan Van Tran Anskar Y. H. Leung Xin Du Qiaoxia Zhang Anh Tun Phan Minh T. N. Le 《Cell proliferation》2022,55(9)
IntroductionAcute Myeloid Leukaemia (AML) is the most common blood cancer in adults. Although 2 out of 3 AML patients go into total remission after chemotherapies and targeted therapies, the disease recurs in 60%–65% of younger adult patients within 3 years after diagnosis with a dramatically decreased survival rate. Therapeutic oligonucleotides are promising treatments under development for AML as they can be designed to silence oncogenes with high specificity and flexibility. However, there are not many well validated approaches for safely and efficiently delivering oligonucleotide drugs. This issue could be resolved by utilizing a new generation of delivery vehicles such as extracellular vesicles (EVs).MethodsIn this study, we harness red blood cell‐derived EVs (RBCEVs) and engineer them via exogenous drug loading and surface functionalization to develop an efficient drug delivery system for AML. Particularly, EVs are designed to target CD33, a common surface marker with elevated expression in AML cells via the conjugation of a CD33‐binding monoclonal antibody onto the EV surface.ResultsThe conjugation of RBCEVs with the CD33‐binding antibody significantly increases the uptake of RBCEVs by CD33‐positive AML cells, but not by CD33‐negative cells. We also load CD33‐targeting RBCEVs with antisense oligonucleotides (ASOs) targeting FLT3‐ITD or miR‐125b, 2 common oncogenes in AML, and demonstrate that the engineered EVs improve leukaemia suppression in in vitro and in vivo models of AML.ConclusionTargeted RBCEVs represent an innovative, efficient, and versatile delivery platform for therapeutic ASOs and can expedite the clinical translation of oligonucleotide drugs for AML treatments by overcoming current obstacles in oligonucleotide delivery.In this study, we harness red blood cell‐derived EVs (RBCEVs) and engineer them with surface functionalization and exogenous drug loading to develop an efficient drug delivery system for AML. Anti‐CD33 antibody was conjugated to RBCEVs using an enzymatic method combined with the streptavidin‐biotin system. We load the antibody conjugated RBCEVs with ASOs targeting FLT3‐ITD or miR‐125b, 2 common oncogenes in AML, and demonstrate that the treatment with engineered EVs improve leukaemia suppression both in vitro and in vivo. 相似文献
39.
Molchanova MA Andriushina VA Savinova TS Stytsenko TS Rodina NV Voĭshvillo NE 《Bioorganicheskaia khimiia》2007,33(3):379-384
A product of microbiological cleavage of the sterols side chain, androsta-1,4-diene-3,17-dione, is toxic for bacteria, in particular, actinobacteria of the genera Mycobacterium and Arthrobacter. Sterols were transformed into androsta-1,4-diene-3,17-dione by culturing the M. neoaurum VKPM An-1656 strain in a high yield, provided that a sorbent was used for elimination of contact between the bacterial cells and the product. Unlike the cholesterol side chain, the more branched chains of phytosterols were cleaved in the presence of M. neoaurum at a high rate only under turbulent stirring of the culture medium, which intensified the formation of hydrocarbonate ion from NaNI3 in situ. 相似文献
40.