Dissociation and reconstitution of bovine seminal RNAase: Construction of a hyperactive hybrid dimer

The quaternary structure of bovine seminal ribonuclease, the only dimeric protein in the superfamily of ribonucleases, is maintained both by noncovalent forces and by two intersubunit disulfides. The available monomeric derivatives of the enzyme may not be reassembled into dimers. They are catalytically active, but do not retain certain properties of the dimeric enzyme, such as: (i) the ability to respond cooperatively to increasing substrate concentrations in the rate-limiting reaction step; and (ii) the antitumor and immunosuppressive actions. In this report we describe the preparation of stable monomers of seminal ribonuclease which can be reassociated into covalent dimers indistinguishable from the native protein. With this procedure a hybrid dimer was constructed, made up of a native subunit associated to a subunit catalytically inactivated by selective alkylation of the active site His-119. This dimer was found to have enzymic properties typical of monomeric ribonucleases, such as a hyperbolic saturation curve in the hydrolytic rate-limiting step of the reaction. However, the hybrid dimer was one order-of-magnitude more active than the dimeric enzyme.     

The increase of hydroxyproline-containing proteins in Jerusalem artichoke mitochondria during the development of cyanide-insensitive respiration

Mitochondria purified from freshly cut and from aged slices of Jerusalem artichoke tubers contain hydroxyproline proteins. These proteins are present in much higher amounts in mitochondria with KCN-insensitive respiration, i.e. in those from aged slices. It is suggested that the dependence of KCN-insensitive respiration on Vit. C is due to the fact that the development of this alternate oxidase requires the ascorbic acid-dependent synthesis of a hydroxyproline-containing mitochondrial protein.     

Structural and functional lability induced by diethyl ether on the sarcoplasmic reticulum membrane

Structural and functional changes occuring in sarcoplasmic reticulum vesicles following exposure to low concentrations (5–7%, v/v) of diethyl ether in aqueous media, were studied by electron microscopy and by kinetic measurements of Ca²⁺ transport and ATPase activity. Electron microscopy of thin sectioned and freeze-fractured sarcoplasmic reticulum vesicles provided detailed resolution of Ca-ATPase amphiphilic molecules displaying ‘lollipop’ portions on the outer surface of the vesicle, and non-polar moieties penetrating the membrane's hydrophobic interior. This asymmetric disposition of ATPase molecules was disrupted in vesicles exposed to ether and then centrifuged and/or resuspended in aqueous media. Such vesicles had a tendency to undergo fragmentation, and the distribution of ATPase molecules was markedly altered. The continuous fuzzy layer of lollipops became discontinuous, and the intramembranous particles became randomly distributed over both the concave and the convex freeze-fracture membrane faces. Functionally, the vesicles lost their ability to accumulate calcium in the presence of ATP, although high rates of ATPase activity were maintained. Vesicles which were simply exposed to ether, without being subjected to centrifugation and/or homogenization, did not appear altered ultrastructurally, and retained their ability to accumulate calcium. In fact, the enzyme turnover and the maximal levels of calcium uptake were increased. It is concluded that diethyl ether interferes with lipid-lipid and protein-lipid interactions in the sarcoplasmic reticulum vesicle membrane, thereby facilitating molecular motions which may be a limiting factor in the transport mechanism. On the other hand, these weakened interactions permit structural denaturation and loss of the ability to maintain a transmembrane Ca²⁺ gradient when the vesicles are subjected to mechanical perturbations which are harmless in the absence of ether.     

The Footprint of Continental-Scale Ocean Currents on the Biogeography of Seaweeds

Explaining spatial patterns of biological organisation remains a central challenge for biogeographic studies. In marine systems, large-scale ocean currents can modify broad-scale biological patterns by simultaneously connecting environmental (e.g. temperature, salinity and nutrients) and biological (e.g. amounts and types of dispersed propagules) properties of adjacent and distant regions. For example, steep environmental gradients and highly variable, disrupted flow should lead to heterogeneity in regional communities and high species turnover. In this study, we investigated the possible imprint of the Leeuwin (LC) and East Australia (EAC) Currents on seaweed communities across ~7,000 km of coastline in temperate Australia. These currents flow poleward along the west and east coasts of Australia, respectively, but have markedly different characteristics. We tested the hypothesis that, regional seaweed communities show serial change in the direction of current flow and that, because the LC is characterised by a weaker temperature gradient and more un-interrupted along-shore flow compared to the EAC, then coasts influenced by the LC have less variable seaweed communities and lower species turnover across regions than the EAC. This hypothesis was supported. We suggest that this pattern is likely caused by a combination of seaweed temperature tolerances and current-driven dispersal. In conclusion, our findings support the idea that the characteristics of continental-scale currents can influence regional community organisation, and that the coupling of ocean currents and marine biological structure is a general feature that transcends taxa and spatial scales.     

ISOLATION OF METAPHASE CHROMOSOMES FROM HELA CELLS

The authors have developed a method for large-scale isolation of metaphase chromosomes from HeLa cells. The distinguishing feature of this method is the use of a pH sufficiently low (about 3) to stabilize the chromosomes against mechanical damage. Many milligrams of fairly pure, morphologically intact chromosomes can be isolated in 8 hr or less of total working time. The isolated chromosomes contain about 2.0 mg of acid-soluble protein, 2.7 mg of acid-insoluble protein and 0.66 mg of RNA for each milligram of DNA. The RNA bound to the isolated chromosomes consists mainly of ribosomal RNA, but there is also a significant amount of 45S RNA.     

Distribution and Numbers of Pygmies in Central African Forests

Pygmy populations occupy a vast territory extending west-to-east along the central African belt from the Congo Basin to Lake Victoria. However, their numbers and actual distribution is not known precisely. Here, we undertake this task by using locational data and population sizes for an unprecedented number of known Pygmy camps and settlements (n = 654) in five of the nine countries where currently distributed. With these data we develop spatial distribution models based on the favourability function, which distinguish areas with favourable environmental conditions from those less suitable for Pygmy presence. Highly favourable areas were significantly explained by presence of tropical forests, and by lower human pressure variables. For documented Pygmy settlements, we use the relationship between observed population sizes and predicted favourability values to estimate the total Pygmy population throughout Central Africa. We estimate that around 920,000 Pygmies (over 60% in DRC) is possible within favourable forest areas in Central Africa. We argue that fragmentation of the existing Pygmy populations, alongside pressure from extractive industries and sometimes conflict with conservation areas, endanger their future. There is an urgent need to inform policies that can mitigate against future external threats to these indigenous peoples’ culture and lifestyles.