The loss of these native dimer interactions weakens the hexameric LysB28ProB29-human insulin complex formed in the presence of phenolic ligands. Thus, it is hypothesized that the diffusion of the phenolic ligands from the site of injection results in the dissociation of hexamers directly to monomers, thereby maintaining the rapid time-action of the monomeric analog in spite of the hexameric conformation in therapeutic formulations.

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We have examined the role of 4-thiouridine in the responses of Salmonella typhimurium to near-UV irradiation. Mutants lacking 4-thiouridine (nuv) and mutants defective in the synthesis of ppGpp (guanosine 5'-diphosphate-3'-diphosphate) (relA) were found to be sensitive to killing by near-UV. Near-UV induced the synthesis of a set of proteins that were not induced in the nuv mutant. Some of these proteins were identified as oxidative defense proteins, and others were identified as ppGpp-inducible proteins. Over 100-fold increases in ApppGpp (adenosine 5', 5"'-triphosphoguanosine-3"'-diphosphate, the adenylylated form of ppGpp) were observed in wild-type cells after near-UV irradiation but not in the 4-thiouridine-deficient mutant. These data support a model in which ppGpp and ApppGpp, a dinucleotide proposed to be synthesized by tRNA-aminoacyl synthetases as a response to the cross-linking of 4-thiouridine in tRNA by near-UV, induce the synthesis of proteins necessary for resistance to near-UV irradiation.One of the most common stresses confronted by organisms is solar radiation. The most energetic part of the solar spectrum which penetrates the atmosphere is the near-UV region (300 to 400 nm) (22). Studies on the environment mortality of bacteria have found that die-off is dependent on light exposure, especially the near-UV portion of the solar spectrum (10,16,17). Since enteric bacteria such as Escherichia coli must adapt to exposures of near-UV during transmission between hosts (8), they are an ideal system for characterizing mechanisms involved in protection from the toxic effects of near-UV.The irradiation of growing E. coli cells with near-UV results in the cessation of growth long before lethal effects are observed (21,22). This growth lag is dependent on the tRNA-modified base 4-thiouridine (s4U), which is found in 65% of tRNA species (22,40,45). This base is unusual in that it has an absorption maximum in the near-UV region (334 nm). After exposure to near-UV, S4U participates in a photochemical reaction which generates intramolecular cross-linking between S4U and a specific cytosine (13,14,29). These cross-linked tRNAs have been shown to be poor substrates for aminoacylation (51). The accumulation of nonaminoacylated tRNA serves to trigger the stringent response via the relA gene product, which is involved in the synthesis of ppGpp (guanosine 5'-diphosphate-3'-diphosphate) (9, 15), which then acts to inhibit stable RNA synthesis while inducing amino acid biosynthesis (15, 43). Inhibition of protein synthesis after near-UV exposure in E. coli is a result of both the stringent response and decreased aminoacylation of tRNA (44).Near-UV exerts toxic effects on E. coli and Salmonella typhimurium at higher fluences, primarily through oxidative mechanisms (22, 23). Macromolecules which absorb in the near-UV region (i.e., heme and flavins) may act as endogenous photosensitizers, transferring the absorbed energy to 02 and forming toxic oxygen species (10,22,23,48). The response of bacteria to oxidative stress (s...

Exposure of cultured mammalian cells to a variety ofconditions that induce the synthesis ofstress proteins, including hyperthermia, ethanol, cadmium, and arsenite resulted in an increased cellular content of adenyl dinucleotides incuding diadenosine tetraphosphate (Ap4A). Exposure to other agents that cause metabolic perturbations not known to induce the synthesis of stress proteins, such as cyclohexamide, cytosine arabinoside, hydroxyurea, and ultraviolet irradiation did not alter the content ofthese nucleotides. It is proposed that these unique nucleotides may mediate adaptive responses of mammalian cells to environmental stress.A variety of metabolic stresses such as hyperthermia (1, 2), heavy metals (2, 3), amino acid analogs (2), uncouplers of oxidative phosphorylation (1), anoxia (4, 5), ethanol (1, 6, 7), and arsenite (5) have been shown to induce a similar stress response in both prokaryotic and eukaryotic cells (reviewed in ref. 8) that has been termed the heat shock response. This response is manifested at several levels of metabolic regulation and includes major changes in both transcription and translation. These changes result in the accelerated synthesis of a group of proteins called heat shock or stress proteins. The synthesis of these proteins is correlated with enhanced cell survival and the induction of cross-protection between different stresses (1-5, 6-15). Ames and coworkers have shown that agents that induce the synthesis of stress proteins in bacterial cells result in an increased cellular content of a unique group of adenyl dinucleotides including diadenosine 5',5'`-P',P4-tetraphosphate (Ap4A) (16,17). We report here that adenyl dinucleotide metabolism is altered in cultured mammalian cells after several different treatments that induce the synthesis of stress proteins. We propose that these unique dinucleotides are involved in adaptive responses of mammalian cells to environmental stress.

An ambitious 10‐year collaborative program is described to invent, design, demonstrate, and support commercialization of integrated biopharmaceutical manufacturing technology intended to transform the industry. Our goal is to enable improved control, robustness, and security of supply, dramatically reduced capital and operating cost, flexibility to supply an extremely diverse and changing portfolio of products in the face of uncertainty and changing demand, and faster product development and supply chain velocity, with sustainable raw materials, components, and energy use. The program is organized into workstreams focused on end‐to‐end control strategy, equipment flexibility, next generation technology, sustainability, and a physical test bed to evaluate and demonstrate the technologies that are developed. The elements of the program are synergistic. For example, process intensification results in cost reduction as well as increased sustainability. Improved robustness leads to less inventory, which improves costs and supply chain velocity. Flexibility allows more products to be consolidated into fewer factories, reduces the need for new facilities, simplifies the acquisition of additional capacity if needed, and reduces changeover time, which improves cost and velocity. The program incorporates both drug substance and drug product manufacturing, but this paper will focus on the drug substance elements of the program.