Laser light-scattering analysis of protoplasmic streaming in the slime mold physarum polycephalum

Laser light scattering is shown to be an effective means of obtaining a rapid, objective assessment of dynamic changes in the intact plasmodium of the myxomycete Physarum polycephalum during bidirectional (shuttle) streaming. The motion of material in a 100 μm diameter region of a plasmodial vein was studied by following changes in the autocorrelation function of the fluctuations in the scattered light intensity. The autocorrelation function was recorded at 10 s intervals and analyzed to follow changes in the flow velocity of protoplasm associated with shuttle streaming. Rhythmic velocity changes and a “beating” pattern of velocity maxima were readily observed. In an attempt to locate the site of underlying structural changes in the vein responsible for the changing pattern of flow, the average scattered intensity was separated into components derived from moving and stationary scatterers. Periodic variations in the light intensity due to stationary scatterers are related to the streaming cycle and indicate the occurrence of important structural changes in the vein walls. Two possible interpretations of the data are offered; one involving gross dynamic changes in vein structure, the other involving the formation, contraction, or breakdown of fibrillar material in the vein wall during the streaming cycle.     

Structure of a β-galactan isolated from the nuclei of Physarum polycephalum

A sulfated and phosphorylated β-D-galactan ([α]_D + 8°) was isolated from the nuclei of the acellular slime mould Physarum polycephalum. The polysaccharide was isolated from cesium chloride gradients during the preparation of ribosomal DNA and purified. The purified galactan contained 89% galactose, 2.5% phosphate and 9.6% sulfate groups and had an average degree of polymerisation of 560. Periodate degradation and permethylation studies indicated the presence of mainly (1 → 4)-, but also of (1 → 3)-, and (1 → 6)-linked galactose units with one branch every 13 units. These results suggested that the intranuclear galactan, apart from its higher sulfate content, is similar to the extra-cellular polysaccharide produced by P. polycephalum.     

Chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum

The chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum has been examined relative to enzymatic activity and reactivity of these groups in the native protein. 4,4′-Dipyridyl disulfide, dansylaziridine, and fluorescein mercuric acetate all reacted with just one of six sulfhydryls per enzyme subunit, resulting in activities of 100, 95 and 70%, respectively. The K_m values for MgATP, formate, and tetrahydrofolate were unaltered in the modified enzymes. ATP did produce a 2.5-fold reduction in the rate of reaction between the enzyme and 4,4′-dipyridyl disulfide. Tetranitromethane reacted most rapidly with a single sulfhydryl group per subunit to produce a 20–30% loss in activity. Subsequent additions of tetranitromethane modified 2.2 tyrosines per subunit which was proportional to the loss of the remaining enzymatic activity. Folic acid, a competitive inhibitor, protected against modification of the tyrosines and the associated activity losses; however, the oxidation of the single sulfhydryl group and the initial 20–30% activity loss were unaffected. In the presence of folic acid, higher concentrations of tetranitromethane produced a loss of the remaining activity proportional to the modification of 1.2 tyrosines per subunit. It is proposed that at least 1 tyrosine critical for enzymatic activity is located at or near the folic acid/tetrahydrofolate binding site.     

Selective modification of positive chemotaxis in the true slime mold Physarum polycephalum by ethylenediaminetetraacetic acid treatment.

The plasmodium of the true slime mold Physarum polycephalum was treated with EDTA or EGTA and the effect of the treatment on the chemotactic response was examined by measuring the chemotactic motive force with the double-chamber method. The results obtained were as follows: (1) The treatment of the plasmodium with 5 mM EDTA (pH 7.0, 20 min) did not give any significant effect on the protoplasmic streaming or motility. (2) The plasmodium treated with EDTA exhibited no chemotactic response to non-electrolyte attractants (D-glucose, D-galactose, D-mannose, and maltose) and negative chemotaxis to electrolyte attractants (cyclic AMP and NaH2PO4). (3) The EDTA treatment gave no effect on the chemotactic response to repellents (D-fructose, NaCl, CaCl2, MgCl2). (4) The EDTA-treated plasmodium exhibited changes in the membrane potential in response to both attractants and repellents as similar to the untreated plasmodium. (5) The treatment of the plasmodium with 5 mM EGTA (pH 7.0, 20 min) gave results similar to those obtained with the EDTA treatment. The results obtained suggested that EDTA (or EGTA) treatment did not affect the receptor sites but modified the transduction mechanism from reception into tactic movement.     

Cyclic AMP in the cell cycle of Physarum polycephalum

Cyclic AMP, [³H]thymidine incorporation, and DNA content were measured in the cell cycle of Physarum polycephalum. A sensitive radioimmunoassay was employed to assay cyclic AMP so that plasmodia could be assayed individually. In contrast to previously published results (Lovely, J.R. and Threlfall, R.J. (1976) Biochem. Biophys. Res. Commun. 71, 789–795), no pre-mitotic peak of cyclic AMP was detected. In seven experiments levels of cyclic AMP showed only small changes in individual experiments and ranged from 1–6 pmol/mg protein in different experiments. When plasmodia in the immediate premitotic period were collected on the basis of nuclear mitotic morphology, no evidence of a peak of cyclic AMP was found. Light was found to increase plasmodial cyclic AMP in a rapid, transient fashion. However, the brief exposure of cell cycle samples to light during collection did not induce any apparent cell cycle specific peaks of cyclic AMP. Although the occurrence of extremely rapid transient peaks of cyclic AMP in the cell cycle cannot be ruled out, it appears likely that the P. polycephalum cell cycle can proceed normally without major changes in cyclic AMP.     

Juvenile hormone-induced biosynthesis of vitellogenin in Leucophaea maderae

Isolated abdomens of virgin female Leucophaea maderae, a South American cockroach, could be induced by a single injection of juvenile hormone I to synthesize vitellogenin. Synthetic capacity was assayed by removal of the fat body at various times after induction and incubation in organ culture with radioactive leucine for 5 h. Under these conditions, active tissues secrete radioactive vitellogenin into the medium after a lag of 2 h and continue secretion at a fixed rate for up to 8 h. Vitellogenin in the tissue reaches a steady-state level after about 3 h. By 5 h, approximately 80% of the newly synthesized vitellogenin can be found in the medium and constitutes 75% of the secreted protein.Vitellogenin does not appear in the medium until 18 h after juvenile hormone induction, rises to a maximum between 72 and 96 h, and has declined to half by 120 h postinduction. At the maximum, about 75% of the total protein secreted is vitellogenin, a more than 50-fold stimulation over injected controls. Since virtually the same quantitative effect of juvenile hormone was found in abdomens with and without ovaries, secondary stimulation by ovarian hormones does not appear to be involved in vitellogenin synthesis in this insect.Oocyte vitellogenin exists as a 560,000 molecular weight monomer complex and a 1,590,000 molecular weight trimer. The smaller complex is only found in young oocytes. By sodium dodecyl sulfate-gel electrophoresis, we find that the 560,000 molecular weight 14S monomer consists of four discrete peptides in the ratio A₁B₁C₂D₂ with molecular weights 118,000, 87,000, 57,500, and 96,000, respectively. The 28S trimer contains only the three peptides A, B, and C in the ratio A₁B₃C₂. These stoichiometries satisfactorily account for the molecular weights of both complexes when corrected for the lipid content. Maturation of 14S to 28S probably involves specific proteolytic conversion of D to B^D, a different peptide of the same size as B, suggesting that the true composition of 28S is A₁B₁B₂^DC₂.The 14S vitellogenin synthesized and secreted by fat body in tissue culture consists mostly of larger polypeptides. There is a major fraction of 179,000 molecular weight, a minor fraction of >260,000 molecular weight, and other smaller polypeptides. Although the exact relationship between these polypeptides and the mature subunits of vitellogenin are not yet clear, it is evident that the multiple protein subunits of this complex protein are synthesized as one or more precursor proteins, followed by proteolytic processing to the mature size.     

Tissue antigens from the third instar larva of Drosophila melanogaster

Antibodies were prepared against the soluble proteins from six tissues of Drosophila larvae. These were used to analyse the antigens in different tissues and at different developmental stages. The results suggest (1) the pattern of antigens determines the characteristics of a tissue, (2) salivary gland antigens are sequestered by the imaginal disks, (3) not all pupal glue antigens are synthesized in the salivary glands, and (4) most larval serum antigens are synthesized by the fat body.     

Comparative studies of the structure and composition of the plasmalemma and the tonoplast in Saccharomyces cerevisiae

The two membranes, plasmalemma and tonoplast (Saccharomyces cerevisiae H 1022), are characterized ultrastructurally by their different texture in the corresponding freeze-fracture faces and their silver staining properties.Biochemical characterization with regard to proteins and lipids indicated that the ratio of protein to lipid is significantly higher in the plasmalemma as compared to the tonoplast. Moreover, a pronounced difference appears to exist for both the amount and the composition of total lipids, phospholipids and sterols. The protein patterns of the plasmalemma and the tonoplast reveal only minor differences, as judged by sodium dodecyl sulphate gel electrophoresis.     

The defensive secretion of the opisthobranch mollusc Onchidella binneyi

Opisthobranch molluscs of the family Onchidiacea have been reported to employ a chemical deterrent as a protection against predators. A single lipid-soluble compound, onchidal, has been isolated from the defensive secretion of Onchidella binneyi. The structure of onchidal was determined from spectral data and from chemical degradation studies.     

Primary structure of the Klebsiella serotype 16 capsular polysaccharide

The primary structure of the Klebsiella serotype 16 capsular polysaccharide consists of tetrasaccharide repeating-units comprising a/ar3)-α-D-Glcp-(1/ar4)β-D-GlcAp-(1/ar4)-α-L-Fucp-(1/ar chain with a β-D-Galp-(1→ branch at position 4 of the D-glucosyl residue.     

Distribution of antigens in established cell lines of Drosophila melanogaster

Antibodies prepared against two Drosophila cell lines together with antibodies prepared against other Drosophila extracts were used to study the distribution of antigens in 10 cell lines. The results suggest: (1) that cultured cells express differentiated functions; (2) that each cell line, including sub-clones of a clone, is unique; and (3) that the cell lines are derived from the lateral ectoderm.     

Cellular and volumetric changes in relation to the activity cycle in the corpora allata of Diploptera punctata

In the corpora allata (CA) of the viviparous cockroach, Diploptera punctata, a cycle of juvenile hormone (JH) synthesis during ovarian maturation can be correlated with cyclical changes in CA volumes and cell numbers. Uptake of [³H]-thymidine occurs in nuclei of CA cells during periods of increase in cell number. Both members of a pair of CA maintain symmetry of volume, cell number and rate of JH synthesis. After a cycle of CA activity, the CA can be transplanted to a young, allatectomized female, where they support a second wave of oöcyte development.     

Chemical composition and ultrastructure of the epicuticular wax in four mutants of Pisum sativum (L)

The action of mutations affecting the epicuticular wax of Pisum sativum has been investigated at the chemical and ultrastructural level. Upper and lower surfaces of the leaves were found to differ markedly in both ultrastructure and chemistry. Mutations affected primarily either the lower (wa, wb and wsp) or the upper surface (wlo), but some effects of all 4 genes could be seen on both surfaces. Specific biochemical lesions could be implied for wsp and wa but the chemical effects of wb and wlo were more diffuse. Generally a close relation between chemical composition and crystallite form of the wax was evident throughout the work.