Identification and Partial Purification of a Stage-Specific 33 kDa Mitochondrial Protein as the Alternative Oxidase of the Trypanosoma brucei brucei Bloodstream Trypomastigotes

ABSTRACT. The glycerophosphate oxidase (GPO), the unique terminal oxidase of bloodstream trypanosome (TAO), appears to be functionally similar to the alternative oxidases of some plants and higher fungi. Immunoblotting of mitochondrial proteins of bloodstream trypomastigotes of Trypanosoma brucei brucei with monoclonal or polyclonal antibodies to Sauromatum guttatum (voodoo lily) and Symplocarpus foetidus (skunk cabbage) alternative oxidases respectively revealed two proteins of about 33 kDa (p33) and 68 kDa (p68). These proteins are not present in procyclic trypomastigotes. Electrophoresis under rigorous denaturing conditions indicated p68 to be the dimer of p33. Indirect immunofluorescent studies of bloodstream and procyclic trypomastigotes with monoclonal antibody to plant alternative oxidase also showed the localization of 33 kDa protein in the mitochondria of the bloodstream trypomastigotes. The functional TAO activity could be solubilized efficiently from the mitochondrial membrane of the bloodstream trypomastigotes by 1% NP-40 or 10 mM lauryl maltoside. When fractionated by Superose 12 gel filtration chromatography, p33 was co-purified with the TAO enzymatic activity. The apparent molecular size of the active enzyme complex was found to be 160 kDa. Gradual disappearance of the 33 kDa protein and the TAO enzymatic activity were well correlated during in vitro differentiation of the bloodstream to procyclic trypomastigotes. This study implies that the net biosynthesis of p33, an essential subunit of TAO, is decreased during differentiation from bloodstream to procyclic trypomastigotes.     

MONOCLONAL ANTIBODY RAISED AGAINST HUMAN MITOTIC CYCLIN B1IDENTIFIES CYCLIN B-LIKE MITOTIC PROTEINS IN SYNCHRONIZED ONION (ALLIUM CEPA L.) ROOT MERISTEM

Cyclin B-like mitotic proteins have been detected in synchronized Allium cepa L. root tip cells by using mouse monoclonal anti-cyclin B₁antibody raised against human cyclin B₁Immunoblot shows two closely placed isoforms of cyclin B-like proteins having an apparent molecular weight around 54 kDa. In vivo [³⁵S]-methionine labelling followed by immunoprecipitation and autoradiography indicates that cyclin B-like proteins are mainly synthesized in the G2 phase of the cell cycle and destroyed in late mitosis. Immunoblotting data depict that the level of cyclin B-like proteins reaches the maximum at the late G2 to early M phase; and it becomes degraded in the late hours of mitosis. Moreover, the cyclin B isoforms are stabilized in colchicine-arrested metaphase cells as already reported in animal cells.     

STUDIES ON TWO APHID-TRANSMITTED VIRUSES OF LEGUMINOUS CROPS

Pea mosaic virus was transmitted by Myzus persicae Sulz., Macrosiphum pisi Kalt., M. solanifolii Ash. and Aphis fabae Scop., but not by Hyperomyzus staphyleae Koch. It is a 'non-persistent' virus (Watson & Roberts, 1939), and is most readily transmitted when vectors are fasted and then given a short infection feeding. Vector efficiency was not increased by increases in preliminary fasting beyond 15 min. or with increasing infection feeding beyond 1 hr. Most aphids became non-infective within 15 min. when feeding, but fasting aphids remained infective for 3 hr. Species that fed readily on the infected plants were less efficient vectors than those which did not. Seed set by infected plants produced healthy seedlings.
Pea enation mosaic virus persisted in Myzus persicae and Macrosiphum pisi for more than 140 hr.; its transmission was unaffected by preliminary treatments of aphids. No transmission was obtained until at least 4 hr. after aphids had left infected plants; usually the 'latent' period exceeded 1 day and its duration was apparently unaffected by the length of the infection feeding.     

THE SPREAD OF VIRUS DISEASES TO SINGLE POTATO PLANTS BY WINGED APHIDS

Young potato plants in pots exposed in the open near plots of potatoes for limited periods at intervals during the summer, became infested with large numbers of winged aphids only during warm, calm and dry weather. Although visited by aphids during May and June, when much of the spread of viruses occurred in nearby potato crops, few of the potted plants became infected. Most potted plants became infected in July when alate aphids were leaving neighbouring potato crops. Widely different proportions of the exposed plants became infected in different years; in two of the three years, many more plants were infected with virus Y than with leaf roll virus.     

SOME PROPERTIES OF BROAD-BEAN MOTTLE VIRUS

A severe disease affecting many plants in a crop of broad beans was found to be caused by a previously undescribed virus, provisionally named broad-bean mottle virus. The distribution of diseased plants suggested spread by a vector, but none of the six insects tested transmitted it. The virus was transmitted to several species of leguminous plants by mechanical inoculation of sap; infectivity for some hosts seemed to be increased by propagation in these hosts.
The virus has an unusual combination of properties. Its thermal inactivation point is about 95°C., whereas sap becomes non-infective within 3 weeks at room temperature. The infection end-point of broad-bean sap is 1/1000, only a little higher than the precipitation titre with specific antiserum. Precipitation with antiserum occurs over a smaller range of antigen/antibody ratios than with other viruses previously studied, possibly because of its greater solubility; it is not precipitated with (NH₄)₂SO₄ until the salt concentration exceeds 75% saturation.
A specific nucleoprotein, containing nucleic acid of the ribose type, can be isolated from infective broad-bean sap in yields up to 2 g./l. Purified preparations, made by salt precipitation and ultracentrifugation, contain uniform spherical particles approximately 17 mμ in diameter. It is suggested that much of this nucleoprotein is non-infective, but may otherwise resemble infective particles.     

Synaptic Vesicle Dynamics: A Simple Model of Phasic Release

We present a simple model of phasic neurotransmitter release whichreproduces the salient features of chemical neurotransmission. The synapticvesicle cycle has been modelled as a set of biochemical reactionsrepresented by a system of coupled differential equations. These equationshave been solved analytically to obtain the time dependent behaviour of thesystem on perturbation from the steady state. The scheme of the synapticvesicle network has been emphasized and its role in determining some of themajor experimentally observed properties of synaptic transmission has beendiscussed, which includes the biphasic decay of the rate neurotransmitterrelease even under sustained stimulation. Another interesting outcome ofthis theoretical exercise is the saturation of total release with thecalcium dependent rate constant. The theoretically calculated values oftotal release fit very well into a sigmoidal saturating function with afourth order cooperativity exponent similar to the empiricalDodge–Rahamimoff equation. It appears that the synaptic vesiclenetwork itself is responsible for some of the major properties associatedwith chemical neurotransmission.