Transbilayer distributions of red cell membrane phospholipids in unilamellar vesicles

The phospholipid organization in unilamellar vesicles comprised of various purified phospholipid components of monkey erythrocyte membrane was ascertained using phospholipase A2 and trinitrobenzenesulfonic acid as external membrane probes. The vesicles were formed by sonication or detergent dialysis and fractionated by centrifugation or gel permeation chromatography. Experiments were done to confirm that the phospholipase A2 treatments did not cause lysis or induce fusion of the vesicles. This enzyme hydrolysed only the glycerophospholipids in the outer surface of the vesicles. The amounts of the external phospholipids determined by this enzymatic method were verified using the chemical probe, trinitrobenzenesulfonic acid. The choline-containing phospholipids and phosphatidylethanolamine localized randomly in the two surfaces of sonicated vesicles (outer diameter, about 30 nm), whereas phosphatidylserine preferentially distributed in the inner monolayer. This phosphatidylserine asymmetry virtually disappeared in detergent dialysed vesicles (outer diameter, about 45 nm). Furthermore, inclusion of cholesterol in both the types of vesicles resulted in more random glycerophospholipid distributions across the plane of vesicles bilayer, presumably due to the cholesterol-induced increases in the size of vesicles. These results demonstrate that the transbilayer distribution of erythrocyte membrane phospholipids in unilamellar vesicles are controlled mainly by the surface curvature rather than by interlipid interactions, and therefore suggest that phospholipid-phospholipid and phospholipid-cholesterol interactions should not play any significant role in determining the membrane phospholipid asymmetry in red cells. It is proposed that this asymmetry primarily originates from differential bindings of phospholipids with membrane proteins in the two leaflets of the membrane bilayer.     

Metalloproteinase inhibitors from bovine cartilage and body fluids

Inhibitors of the mammalian metalloproteinases, collagenase, proteoglycanase and gelatinase were isolated from bovine cartilage (extracts and culture medium) and bovine amniotic fluid and serum. These inhibitors either bind or do not bind to concanavalin-A--Sepharose, with Mr (gel filtration) of about 30 000 and 20 000, respectively. Cartilage and chondrocyte culture media contained only concanavalin-A-binding inhibitors whereas cartilage extracts contained only a non-binding inhibitor: serum and amniotic fluid contained both forms of inhibitory activities. In moist biochemical respects, particularly in their abilities to inhibit metalloproteinases, all of the inhibitors were found to be similar. It is concluded that the forms of the inhibitors that differ in Mr may be closely related to the tissue inhibitor of metalloproteinases (TIMP) previously purified from rabbit and human sources. These findings help to clarify other studies on collagenase inhibitors and support the concept that TIMP-like inhibitors may be important in the control of connective tissue degradation.     

Target antigens in malaria transmission blocking immunity

Malaria transmission blocking immunity has been found to operate against two distinct phases of development of malaria parasites in the mosquito midgut: (i) against the extracellular gametes and newly fertilized zygotes shortly after ingestion by a mosquito of parasitized blood and (ii) against the zygotes during their subsequent development into ookinetes. Immunity is antibody-mediated and stage-specific. A set of three proteins, synthesized in the gametocytes, expressed on the surface of the gametes and newly fertilized zygotes and subsequently shed during later transformation of the zygotes, has been identified as the target antigens of anti-gamete fertilization blocking antibodies. A single protein, synthesized and expressed on the zygote surface during its development to ookinetes, has been identified as the target of antibodies which block the development of the fertilized parasites in the mosquito. Immunization of human populations against gamete or zygote antigens, while not directly protecting an immunized individual from inflection, would reduce the transfer of malaria within the population. Such immunity, in addition to reducing the overall rate of malaria transmission, would, if combined with a vaccine against the asexual (disease-causing) stages, reduce the chance of selection of parasites that are resistant to the asexual vaccine by preventing their entry into the mosquito population.     

Partial purification of a competence factor from Rhizobium japonicum

A competence factor (CF) from Rhizobium japonicum was partially purified to 43 fold on Sephadex G-100. This CF preparation was sensitive to heat, trypsin and pronase, was resistant to DNase 1, RNase A and lysozyme. It had an approximate mol. wt. of 82,000. Osmotic shock treatment of competent cells revealed that the CF is located in the periplasmic region of the cell.Abbreviations CF competence factor - BSA bovine serum albumin - YM yeast mannitol medium     

Axial ranking of residues in collagen in relation to edge association and fibril formation

In our earlier analysis of intermolecular interactions between collagen molecules, a major concern with the program employed is that it compared numbers of interactions between residues located on edges of defined, identical width and thus would not necessarily compare the same number of residues in each edge. This would be particularly true of some values of θ where well-defined vertical ranking of residues occurs. We have examined ranking of residues in relation to intermolecular edge association between bovine skin [α1(I)]₃ model collagen molecules by utilizing two different methods of counting intermolecular interactions between residues. The interaction peaks at θ = 27.69° and 36.00° are absent or relatively less intense in the plots obtained by utilizing radial distances between interacting residues instead of vertical bands of defined width. These studies suggest caution in accepting recently reported analyses of superhelix coiling of the collagen molecule which point to values of 27.69° or 36.00° for the twist of the superhelix. Although intramolecular interactions clearly point to interaction of collagen molecules at D intervals, they are insufficiently restricted in distribution to provide a reliable estimate of the superhelix angle by procedures so far employed.     

Regulation of glutamine synthetase I fromRhizobium meliloti

Glutamine synthetase I fromRhizobium meliloti was found to be inhibited by adenosine 5-monophosphate, alanine, glycine, carbamyl phosphate, cytidine 5-triphosphate, tryptophan, histidine, and glucosamine-6-phosphate. Each inhibitor was independent in its action and the effect was cumulative when more than one inhibitor was added.     

Studies on transducing phage M-1 for Rhizobium japonicum D211

Phage M-1 produced clear plaques with a halo in the lawn of Rhizobium japonicum D211. A one step growth curve of phage M-1 showed a latent period of 3 h, burst size of 55 and rise period of 2 h. The inactivation of phage M-1 was found to be dependent upon the concentraion of d-glucosomanine. The neutralization kinetics of phage M-1 by antiphage serum gave a K value (velocity constant) of 83.1 min^–1. Transduction of str and kan was studied in the presence of antiphage serum and d-glucosamine. Cotransduction of different antibiotic resistance markers suggested that the system can be further explored for high resolution mapping in R. japonicum.Abbreviations YM yeast mannitol medium - PFU plaque forming unit - moi multiplicity of infection - EOP efficiency of plating     

Synergistic effect of heat-shock and UV-irradiation on mitosis and protein synthesis in G2-phase plasmodia of Physarum polycephalum Schw--reduction in UV-induced mitotic delay in a preheat-shocked system

The synergistic effect of UV irradiation and heat-shock during the last 3 hr of G2 phase of the cell cycle in the plasmodia of P. polycephalum, in terms of mitotic delay and inhibition of protein synthesis, has been evaluated. The mitotic delay due to both perturbers coordinately increased closer to mitosis. Maximum mitotic delay was obtained in plasmodia heat-shocked after UV irradiation, indicating the presence in this system of either a heat-labile mitogenic substance which is comparatively less susceptible to UV or a substance which is made more susceptible to hyperthermia by UV. A protective role for heat-shock applied before irradiation has been observed in that, radiation-induced mitotic delay is significantly reduced in this combination. There was severe inhibition of translation in all the perturbed classes. Organelle level effects which are independent of major protein synthetic activities or different levels of heat-shock protein production could be the reason for the lack of correlation between percentage inhibition of general protein synthesis and the extent of mitotic delay with respect to the two double-perturbed systems.