A method for isolation of large, translationally active RNA species is presented. The procedure involves homogenization of cells or tissues in 5 M guanidine monothiocyanate followed by direct precipitation of RNA from the guanidinium by 4 M LiCl. Modifications are described for use with tissue culture cells, yeast, tissues, or isolated nuclei. The advantages of the procedure include speed, simplicity, avoidance of an ultracentrifugation, and its applicability to large numbers of small samples. The procedure yields large mRNA precursors up to 10 kb and mRNA species which translate very well. However, small (less than 300 nucleotides) RNA species are recovered with a poor yield.

The cheW gene from Escherichia coli has been cloned behind an inducible promoter, and the effects of the overproduction of the CheW protein on chemotactic behavior and receptor covalent modification have been examined. Plasmids that contain the cheW gene behind a regulatable promoter complement a cheW mutation when the CheW protein is produced at low levels. However, when the CheW protein is greatly overproduced in either a wild-type strain or a cheW mutant, chemotaxis is greatly inhibited. cheW null mutant cells swim smoothly as if they were constantly responding to an attractant. Surprisingly, cells in which the CheW protein is overproduced also swim smoothly. The behavioral defect produced by overproduction of the CheW protein does not require the presence of the cheR, cheB, or cheZ gene. Receptor demethylation is also inhibited by overproduction of the CheW protein, as it is by a mutation in the cheW gene or a response to an attractant. In all respects, therefore, overproduction of the CheW protein has the same consequences as does a mutation in the cheW gene or a response to an attractant. A model involving two states of the CheW protein is proposed to explain its role in bacterial chemotaxis.Bacterial chemotaxis is an ideal system for the study of the biochemistry and genetics of behavior. The powers of classical bacterial genetics and modem molecular biology have been employed to identify the components of chemotaxis and their functions. The availability of large quantities of purified chemotaxis proteins has permitted the exploration of the biochemical relationships between ti em. Now, as rapid progress in the elucidation of the mechanism of chemotactic signaling is being made, general principles of the biochemistry of excitation, memory, adaptation, and signal amplification and integration are emerging.Extensive progress has been made in understanding how motile bacteria such as Escherichia coli are able to sense gradients of nutrients such as sugars, amino acids, and oxygen and are able to accumulate in regions where these nutrients are in highest concentrations (1,23,24). Peritrichous bacteria have been shown to exhibit two modes of swimming, smooth swimming and tumbling (5, 24). In the absence of a change in the concentration of a chemical stimulant, the bacteria alternate between the two modes and perform random walks (5). When presented with a rapid increase in the concentration of an attractant, such as aspartate or serine, that binds to a specific transmembrane chemotaxis receptor, the cells swim smoothly. After a period of seconds to minutes, depending on the size of the concentration increase, the cells adapt to the presence of the attractant and resume their random walks (24). This adaptation results from a change in the level of receptor methylesterification. Increases in attractant concentrations cause an increase in receptor methylesterification, while decreases in attractant concentrations cause a decrease in receptor methylesterification (34, 36). Methylesterification of multiple specific...

Type 1 diabetes (T1D) is caused by the immune-mediated loss of pancreatic beta cells that produce insulin. The latest advances in stem cell (SC)-beta cell differentiation methods have made a cell replacement therapy for T1D feasible. However, recurring autoimmunity would rapidly destroy transplanted SC-beta cells. A promising strategy to overcome immune rejection is to genetically engineer SC-beta cells. We previously identified Renalase (Rnls) as a novel target for beta cell protection. Here we show that Rnls deletion endows beta cells with the capacity to modulate the metabolism and function of immune cells within the local graft microenvironment. We used flow cytometry and single-cell RNA sequencing to characterize beta cell graft-infiltrating immune cells in a mouse model for T1D. Loss of Rnls within transplanted beta cells affected both the composition and the transcriptional profile of infiltrating immune cells in favor of an anti-inflammatory profile with decreased antigen presenting capacity. We propose that changes in beta cell metabolism mediate local immune regulation and that this feature could be exploited for therapeutic goals.

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Basal lamina (BL) of Torpedo, Discopyge and Electrophorus electric organs was purified in order to establish polypeptide composition and association with acetylcholinesterase (AChE). Results indicate that BL presents a distinct peptide pattern and that the A12 form of AChE is directly attached to it. Comparison of the species studied demonstrated similarities both in polypeptide composition and AChE content of the purified BL. Extractions of BL with solutions of high ionic strength, guanidine-HCl and acetic acid indicated the differential solubilization of various domains of BL polypeptides.