A rapid chitin synthase preparation for the assay of potential fungicides and insecticides

Summary A rapid technique for the preparation of large quantities of solubilized chitin synthase is described. The enzyme derived is very stable and has high specific and total activities. It has been used to screen compounds with potential fungicidal and insecticidal properties.     

Studies on the mechanism of assembly of tobacco mosaic virus.

Sedimentation and proton binding studies on the endothermic self-association of tobacco mosaic virus (TMV) protein indicate that the so-called "20S" sedimenting protein is an interaction system involving at least the 34-subunit two-turn yield cylindrical disk aggregate and the 49-subunit three-turn helical rod. The pH dependence of this overall equilibrium suggests that disk formation is proton-linked through the binding of protons to the two-turn helix which is not present as significant concentrations near pH 7. There is a temperature-induced intramolecular conformation change in the protein leading to a difference spectrum which is complete in 5 x 10(-6) s at pH 7 and 20 degrees C and is dominated at 300 nm by tryptophan residues. Kinetics measurements of protein polymerization, from 10(-6) to 10(3) s, reveal three relaxation processes at pH 7.0, 20 degrees C, 0.10 M ionic strength K (H) PO4. The fastest relaxation time is a few milliseconds and represents reactions within the 4S protein distribution. The second fastest relaxation is 50-100 x 10(-3) s and represents elementary polymerization steps involved in the formation of the approximately 20 S protein. Analysis of the slowest relaxation, approximately 5 x 10(4) s, suggests that this very slow formation of approximately 20 S protein may be dominated by some first order process in the overall dissociation of approximately 20S protein. Sedimentation measurements of the rate of TMV reconstitution, under the same conditions, show by direct measurements of 4S and approximately 20S incorporation at various 4S to approximately 20S weight ratios that the relative rate of approximately 20S incorporation decreases almost linearly, from 0 to 50% 4S. There appears to be one or more regions of TMV-RNA, approximately 1-1.5 kilobases long, which incorporates approximately 20S protein exclusively. Solutions of approximately 95-100% approximately 20S protein have been prepared for the first time and used for reconstitution with RNA. Such protein solutions yield full size TMV, but at a slower rate than if 4S protein is added. Thus the elongation reaction in TMV assembly, following nucleation with approximately 20S protein, is not exclusively dependent upon the presence of either 4S or approximately 20S protein aggregates. The initial, maximum, rate of reconstitution increases about threefold when the protein composition is changed from 5% to 30% 4S protein, at constant total protein concentration at pH 7.0, 20 degrees C in 0.10 M ionic strength K (H)PO4. The probable binding frame at the internal assembly nucleation site of TMV-RNA has been determined by measuring the association constants for the binding of various trinucleoside diphosphates to helical TMV protein rods. The -CAG-AAG-AAG-sequence at the nucleation site is capable of providing at least 10-14 kcal/mol of sites of binding free energy for the nucleation event in TMV self-assembly.     

Transformed cells have lost control of ribosome number through their growth cycle.

Previous studies on the synthesis and function of the protein synthetic machinery through the growth cycle of normal cultured hamster embryo fibroblasts (HA) were extended here to a series of four different clonal lines of polyoma virus-transformed HA cells. Under our culture conditions, these transformed cells could enter a stationary phase characterized by no mitotic cells, very low rates of DNA synthesis, and arrest in a post-mitotic pre-DNA synthetic state. Cellular viability was initially high in stationary phase but, unlike normal cells, transformed cells slowly lost viability. The rate of protein synthesis in the stationary phase of the transformed cells fell to 25-30% of the exponential rate. Though this reduction was similar to that seen in normal cells, it was accomplished by different means. The specific reduction in the ribosome complement per cell to values below that of any cycling cell seen in normal cells, was not seen in any of the transformed lines. This observation, which implies a loss of normal control of ribisome synthesis through the growth cycle after transformation, was confirmed in normal Chinese hamster embryo fibroblasts and transformed CHO cell lines. Normal control of ribosome synthesis was restored in L-73 and LR-73, growth control revertants of one of the transformed CHO lines. The transformed lines reduced their protein synthetic rates in stationary phase either by a greater reduction in the proportion of functioning ribosomes than that seen in normal cells or by a decrease in the elongation rate of functioning ribosomes; the latter effect was not seen in the normal cells. A model for growth control of normal cells and its derangement in transformed cells is presented.     

Use of recombinant plasmids to characterize collagen RNAs in normal and transformed chick embryo fibroblasts.

Two recombinant plasmids containing chick collagen DNA sequences have been used to characterize messenger RNAs for pro-alpha1 (type I) and pro-alpha2 collagen. Poly(A)-containing RNA from chick embryo calvaria and long bones, tissues which are very active in collagen synthesis, were electrophoresed on agarose gels containing methylmercuric hydroxide and transferred to diazobenzyloxymethyl paper; these covalently bound RNAs were hybridized to 32P-labeled pro-alpha1 or pro-alpha2 collagen DNA sequences derived from the recombinant plasmids. The pro-alpha1 collagen probe identified two RNAs, a major species of 5000 bases and a minor species of 7100 bases; the pro-alpha2 collagen probe hybridized to a major species very similar in size to the pro-alpha1 mRNA, about 5200 bases, and a minor species of 5700 bases. It is possible that the 7100 and 5700 base RNAs represent precursors of pro-alpha1 and pro-alpha2 collagen mRNA, respectively. When similar hybridization experiments were performed with RNA from chick embryo fibroblasts, both the pro-alpha1 and pro-alpha2 collagen mRNAs were observed, as well as their corresponding larger species. With RNAs from fibroblasts transformed by Rous sarcoma virus, however, the levels of all RNA species which hybridized with the pro-alpha1 and pro-alpha2 collagen DNA probes were significantly reduced.     

THE ORIGINS OF ADAPTIVE INTERSPECIFIC TERRITORIALISM

1. In order to understand fully the evolution of a behavioural trait one must not only consider whether it is adaptive in its present environment but also whether it originated as an adaptation to existing selective forces or as a fortuitous consequence of selection for a different role in other environments (i.e., as a pre-adaptation) or of selection for different traits (e.g., as a pleiotropic effect). In this paper interspecific territorialism is examined in species of humming-birds, sun-birds, tropical reef fishes, stingless bees, stomatopods, crayfish, and limpets as a means of determining its adaptiveness and its origins. 2. Humming-birds form complex assemblages with species sorted out among the available resources. Dominant species establish feeding territories where flowers provide sufficient nectar. A few large, dominant species, usually uncommon, are marauders on others' territories. Subordinate species establish territories where flowers are more dispersed or produce less nectar, or they fly a circuit from nectar source to nectar source when flowers are even more dispersed, a foraging pattern called ‘traplining’, or they steal nectar from the territorial species by being inconspicuous while foraging. Two species, Amazilia saucerottei and Selasphorus sasin, subordinate in one-to-one encounters, are able to take over rich resources by establishing several small territories within a territory of a dominant and forcing it to forage elsewhere. 3. Among humming-birds, territorial individuals attacked not only subordinate competitors but marauding humming-birds and some insects, which stayed in the territory and foraged at will, and seemingly inappropriate targets, such as non-competitors. This suggests that the stimulus for aggression is ‘any flying organism near the food resources’, regardless of its appearance. The behaviour rather than the identity of the intruder is the stimulus. 4. Sun-birds resemble humming-birds to the extent that dominants establish territories on rich nectar sources and subordinates establish territories on less rich nectar sources or steal from the territories of dominants. The diversity of foraging patterns is not so great as in humming-birds, perhaps because so few species of sun-birds have been studied. However, the advantage of territorialism has been measured in the sun-bird Nectarinia reichenowi. Individuals with territories lose much less nectar to competitors than do those without territories. 5. Field work on three species of tropical reef fishes involved a single aggressive species whose individuals attacked a wide range of species intruding on their territories. The stimulus for aggression in Pomacentrus jenkinsi seemed to be an “object moving through [its] territory”. As suggested for humming-birds, the stimulus is the behaviour rather than the identity of the intruder. 6. The relationships found in stingless bees, stomatopods, crayfish, and limpets are simpler. The dominant and subordinate species divide the resources in their habitat, the dominants' aggression preventing the subordinates from using resources that were otherwise available to them. 7. A general pattern emerges. Mutual interspecific territorialism occurs between species that (i) have different geographic ranges, (ii) occupy different habitats, or (iii) use different resources within the same habitat. Examples of two species holding separate territories on the same resources within the same habitat are rare and occur when the dominant species is rare relative to the available resources. These observations are contrary to the usual view that interspecific territorialism is an adaptation that permits co-existence of potential competitors within the same habitat. 8. Interspecific territorialism is sometimes adaptive and sometimes maladaptive, depending upon the species and the situation. 9. The general pattern of occurrence of the behaviour and the general nature of the stimulus for aggression, i.e., the behaviour rather than the identity of the intruder, suggest that interspecific territoriality is a fortuitous consequence of selection for intraspecific territorialism, the latter being not only an adaptation to the presence of conspecific competitors but a pre-adaptation to the presence of competitors of other species, should they occur.     

An automated version of the periodate-thiobarbituric acid assay for analysis of delta-4,5 unsaturated uronic acids and its application to the assay of hyaluronic acids and chondroitin sulfates

An automated periodate-thiobarbituric acid assay of Δ-4,5 unsaturated uronic acids which avoids extraction of chromogen has been developed and applied to the analysis of hyaluronic acid and chondroitin sulfates in standard glycosaminoglycan mixtures and in biological samples following digestion with eliminase enzymes. Assay of hyaluronic acid was linear between 0.1 and 2.5 μg of uronic acid, when digested with hyaluronidase from S. hyalurolyticus and use directly in the automated procedure. The measurement of unsaturated disaccharide standards (25–100 μm) derived from chondroitin sulfates was also linear although the color yields were different. The proportions of chondroitin sulfate isomers were estimated by assay of the unsaturated chondroitin disaccharides which has been separated by thin-layer chromatography.     

Leaf pretreatment with senescence retardants as a basis for oat protoplast improvement

Protoplasts obtained from oat leaves floated on buffer for 18hr show high nuclease activity, low rates of incorporation ofamino acids and nucleosides into macromolecules, and high ratesof spontaneous lysis. Addition to the leaf flotation mediumof the senescence retardants cycloheximide or kinetin, of thedibasic amino acids L-lysine or L-arginine, or of the diaminesputrescine or cadaverine reduces the rise in nuclease activityand spontaneous lysis of protoplasts, and increases the rateor extent of presumptive protein and nucleic acid synthesis.The diamines, which also retard chlorophyll degradation in theexcised leaves, appear to act both on the membrane and on systemscontrolling macromolecular synthesis and breakdown. By contrast,the senescence promoter L-serine hastens chlorophyll degradationfrom excised leaves and does not improve protoplasts derivedfrom those leaves. (Received July 4, 1977; )     

Immunologic responses to Candida albicans. III. Effects of passive transfer of lymphoid cells or serum on murine candidiasis

Passive transfer of immune serum gave a significant degree of protection against deep seated candidiasis in mice. Repeated attempts to transfer resistance by the transfer of sensitized lymphoid cells gave negative results, even though cutaneous delayed hypersensitivity was transferred by the cells. The results suggest that cell-mediated immunity is not of primary importance in this model of murine candidiasis, and that humoral immunity contributes to protection.