Laterobasal membranes from intestinal epithelial cells: Isolation free of intracellular membrane contaminants

Summary A simplified method for isolating highly purified laterobasal membranes (LBM) from enterocytes is based on treatment of membranes with 8mm CaCl₂ concentration in order to aggregate intracellular membrane contaminants. The resultant LBM showed an average 15-fold enrichment and constituted 8% of the original K-stimulated phosphatase in the initial crude homogenate. It showed typical LBM migration on counter-current distribution (CCD) and was essentially free of contamination with endoplasmic reticulum and Golgi membranes. This method is highly efficient and yields sufficient purified LBM to allow comprehensive analysis of enterocyte membrane events.     

Sexual and developmental differences in the protein pattern of haemolymph and body tissues of Streptocephalus dichotomus Baird (Crustacea: Anostraca)

The protein pattern of haemolymph and body tissues of the freshwater fairy shrimp Streptocephalus dichotomus has been investigated in both sexes, using polyacrylamide disc gel electrophoresis. The electropherograms of four developmental stages show variations in number and intensities of protein fractions. In Stage III, two female-specific proteins of glycolipoprotein nature appear. This stage corresponds to maturity: females begin to possess mature oocytes in the ovary. These two vitellogenic proteins are well represented in the female haemolymph, ovary and freshly laid eggs, but are absent in the male haemolymph. A heterosynthetic mode of yolk formation is thus evident in this anostracan. The two sex-limited proteins are only faintly represented in shelled eggs, suggesting an early utilization of these compounds in embryogenesis.     

Tobacco embryogenesis: Storage-protein-accumulating cells of embryo, suspensor, and endosperm are able to undergo cytokinesis

Summary It is widely accepted that seed storage proteins accumulate only in cells which have entered the cell expansion phase and do not continue to divide. Here we present data indicating that the accumulation of storage globulins in tobacco begins already during early embryogenesis in a period of sustained mitotic activity. Western blot analysis revealed that polypeptides of the legumin-like 12S globulins (M_r 60000, 40000, 20000) appear at mid/late globular stage, whereas the vicilin-like 7S globulin (M_r 50000) follows during the transition from heart to torpedo stage. The accumulation of legumin-like polypeptides begins first in the endosperm during the mid globular stage followed in the embryo-suspensor complex during the heart-shaped stage. The vicilin-related fraction appears first in the endosperm and three days later in the embryo. Examination of individual cells from squash preparations revealed that protein bodies are not confined to intermitotic cells, but are also present in cells undergoing mitosis. Protein bodies of dividing cells situated outside the mitotic apparatus are not metabolized during cytokinesis. The only cell type which loses its protein bodies completely prior to the first mitotic division is the primary hypophysis cell. Our finding that storage proteins can occur in dividing cells independent of their origin and developmental capacity indicates that the cell expansion hypothesis of storage protein accumulation has to be revised.     

Intercompany Energy Integration

Reusing heat through process integration in heat exchanger networks has long been a key measure for increasing energy efficiency in energy‐intensive industries. Thermal pinch analysis is commonly used for a systematic matching of process streams and thus planning of optimal process integration in large chemical plants. The possible savings increase with the amount of heat and the number of integrated process streams. Therefore co‐ siting of several companies in a symbiotic network opens new opportunities for process integration even in small and medium‐size enterprises (SMEs), but also introduces new challenges. Thermal pinch analysis is extended here to account for piping distances and fluctuations and limited availability of energy flows by adding additional costs for the piping system and a backup utility system in the optimization function. Cooperative game theory is proposed to derive a sharing of savings between the partners of the industrial symbiosis that is optimal for each partner and should prevent partners from leaving the network because of higher benefits in a subgroup or alone. It is argued that knowledge about the optimality of a network for each partner creates trust between the partners that is a necessary base for the long‐term commitment needed in industrial symbioses. An exemplary symbiotic network combining the production of pulp and woody biomass energy carriers is used to illustrate the proposed approaches.     

Reduced levels of oxidized and glycoxidized proteins in human fibroblasts exposed to repeated mild heat shock during serial passaging in vitro

Repeated mild heat shock (RMHS) has beneficial hormesis-like effects on various characteristics of human skin fibroblasts undergoing replicative senescence in vitro. We have tested whether RMHS could reduce the accumulation of oxidized and glycoxidized proteins, which is a major age-related change. Levels of carbonylated proteins, furosine, N-carboxymethyl-lysine-rich proteins and advanced glycation end products increased during serial passaging of fibroblasts in culture. However, the extent of accumulation of oxidized and glycoxidized proteins was significantly reduced in RMHS cells. The basal concentration of reduced glutathione was higher and that of oxidized glutathione was lower in RMHS cells. Whereas the basal level of heat shock protein HSP27 decreased in both RMHS and control cells during serial passaging, the increase of the basal level of HSP70 with increasing passage level was significantly higher in RMHS cells. These results show that the slower accumulation of damaged proteins in fibroblasts exposed to RMHS results partly from the increased ability of these cells to cope with oxidative stress, and to synthesize HSP responsible for protein capping and refolding.     

Novel, rapid purification of the membrane protein photosystem I by high-performance liquid chromatography on porous materials

New porous materials have been tested for their potential to speed up purification of membrane proteins. As an example the purification of photosystem I, a light-driven electron pump from the cyanobacterium Synechocystis PCC6803, was optimized. The combination of two HPLC steps (an anion-exchange chromatography followed by a hydrophobic interaction chromatography) yields homogeneous monomeric or trimeric photosystem I as determined by gel filtration and gel electrophoresis. In comparison to traditional purification schemes our method is at least three-times faster and allows for easy scale-up.     

MSI4/FVE is required for accumulation of 24-nt siRNAs and DNA methylation at a subset of target regions of RNA-directed DNA methylation

DNA methylation is an important epigenetic mark. In plants, de novo DNA methylation occurs mainly through the RNA-directed DNA methylation (RdDM) pathway. Researchers have previously inferred that a flowering regulator, MULTICOPY SUPPRESSOR OF IRA1 4 (MSI4)/FVE, is involved in non-CG methylation at several RdDM targets, suggesting a role of FVE in RdDM. However, whether and how FVE affects RdDM genome-wide is not known. Here, we report that FVE is required for DNA methylation at thousands of RdDM target regions. In addition, dysfunction of FVE significantly reduces 24-nucleotide siRNA accumulation that is dependent on factors downstream in the RdDM pathway. By using chromatin immunoprecipitation and sequencing (ChIP-seq), we show that FVE directly binds to FVE-dependent 24-nucleotide siRNA cluster regions. Our results also indicate that FVE may function in RdDM by physically interacting with RDM15, a downstream factor in the RdDM pathway. Our study has therefore revealed that FVE, by associating with RDM15, directly regulates DNA methylation and siRNA accumulation at a subset of RdDM targets.     

Aromatic-aromatic interactions: analysis of π-π interactions in interleukins and TNF proteins

Aromatic-aromatic hydrogen bonds are important in many areas of chemistry, biology and materials science. In this study we have analyzed the roles played by the π-π interactions in interleukins (ILs) and tumor necrosis factor (TNF) proteins. Majority of π-π interacting residues are conserved in ILs and TNF proteins. The accessible surface area calculations in these proteins reveal that these interactions might be important in stabilizing the inner core regions of these proteins. In addition to π-π interactions, the aromatic residues also form π-networks in ILs and TNF proteins. The results obtained in the present study indicate that π-π interactions and π-π networks play important roles in the structural stability of ILs and TNF proteins.     

Molecular and functional analysis of the ribosomal L11 and S12 protein genes ( rplK and rpsL ) of Streptomyces coelicolor A3(2)

A RelC deletion mutant, KO-100, of Streptomyces coelicolor A3(2) has been isolated from a collection of spontaneous thiostrepton-resistant mutants. KO-100 grows as vigorously as the parent strain and possesses a 6-bp deletion within the rplK, previously termed relC. When the wild-type rplK gene was propagated on a low-copy-number vector in mutant KO-100, the ability to produce ppGpp, actinorhodin and undecylprodigiosin, which had been lost in the RelC mutant, was completely restored. Allele replacement by gene homogenotization demonstrated that the RelC mutation is responsible for the resistance to thiostrepton and the inactivation of ppGpp, actinorhodin and undecylprodigiosin production. Western blotting showed that ribosomes from the RelC mutant KO-100 contain only one-eighth the amount of L11 protein found in ribosomes of the parent strain. The impairment of antibiotic production in KO-100 could be rescued by the introduction of mutations that confer resistance to streptomycin (str), which result in alteration of Lys-88 in ribosomal protein S12 to Glu or Arg. No accompanying restoration of ppGpp synthesis was detected in these RelC str double mutants. Received: 12 May 1997 / Accepted: 22 July 1997