Temperature-regulated expression of the tac/lacl system for overproduction of a fungal xylanase in Escherichia coli

Temperature-regulated expression of recombinant proteins in the tac promoter (Ptac) system was investigated. Expression levels of fungal xylanase and cellulase from N. patriciarum in E. coli strains containing the natural lacI gene under the control of the Ptac markedly increased with increasing cultivation temperature in the absence of a chemical inducer. The specific activities (units per milligram protein of crude enzyme) of the fungal xylanase and cellulase produced from recombinant E. coli strain pop2136 grown at 42 degrees C were about 4.5 times higher than those of the cells grown at 23 degrees C and were even slightly higher when compared with cells grown in the presence of the inducer isopropyl beta-D-thiogalactopyranoside. The xylanase expression level in the temperature-regulated Ptac system was about 35% of total cellular protein. However, this system can not be applied to E. coli strains containing lacIq, which confers over production of the lac repressor, for high-level expression of recombinant proteins. In comparison with the lambda PL system, the Ptac-based xylanase plasmid in E. coli pop2136 gave a considerably higher specific activity of the xylanase than did the best lambda PL-based construct using the same thermal induction procedure. The high-level expression of the xylanase using the temperature-regulated Ptac system was also obtained in 10-litre fermentation studies using a fed-batch process. These results unambiguously demonstrated that the temperature-modulated Ptac system can be used for overproduction of some non-toxic recombinant proteins.     

Development of novel, potent, and selective dopamine reuptake inhibitors through alteration of the piperazine ring of 1-[2-(diphenylmethoxy)ethyl]-and 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazines (GBR 12935 and GBR 12909)

The design, synthesis, and biological evaluation of compounds related to the dopamine (DA) uptake inhibitors: 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine (1) and 1-[2-[bis-(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (2) (GBR 12395 and GBR 12909, respectively), directed toward the development and identification of new ligands interacting with high potency and selectivity at the dopamine transporter (DAT) is reported. The substitution of the piperazine ring in the GBR structure with other diamine moieties resulted in the retention of the high affinity of new ligands for the DAT. Some of the modified GBR analogs (e.g. 8, 10, (-)-49, or (-)-50) displayed substantially higher selectivity (4736- to 693-fold) for the dopamine (DA) versus the serotonin (5HT) reuptake site than the parent compounds. The bis(p-fluoro) substitution in the (diphenylmethoxy)ethyl fragment slightly increased the affinity of the ligands at the DA reuptake site but reduced their selectivity at this site (e.g. 9 and 8, 11 and 10, or 17 and 16, respectively). Congeners, such as the series of monosubstituted and symmetrically disubstituted piperazines and trans-2,5-dimethylpiperazines, which lack the (diphenylmethoxy)ethyl substituent lost the affinity for the DAT yet exhibited very high potency for binding to the sigma receptors (e.g.28). The chiral pyrrolidine derivatives of 1, (-)-49, and (+)-49, exhibited an enantioselectivity ratio of 181 and 146 for the inhibition of DA reuptake and binding to the DAT, respectively.     

Antioxidant activity in leaves of some mediterranean plants

Hexane and methanol leaf extracts of sixteen Mediterranean plant species were obtained by percolation. Higher yields were observed for methanol (16.4–47.8%) than for hexane extracts (1.3–13%). Antioxidant activity of these extracts was determined by a rapid spectrophotometric method involving the combined oxidation of beta-carotene and linoleic acid. Leaf extracts were thus characterized by an Antioxidant Activity Coefficient (AAC) ranging from 0 to 1000. Hexane extracts gave much higher AAC values than the corresponding methanol extracts. In both cases, myrtle (Myrtus communis) showed the best antioxidant effectiveness.     

Glutarimide acrylic copolymer: A new route to high heat PVC

PVC is the second largest polymer produced and used in the U.S. It is a self-extinguishing material with excellent chemical resistance and good dimensional stability. It's greatest asset, however, is it's versatility. By specifically formulating PVC with the numerous types of additives available, a balance of properties can be tailored for a wide range of applications. The most serious disadvantage of PVC is it's low heat resistance which limits the service temperature of finished articles to about 70°C. Now an additive is available that can be formulated with PVC to form miscible, transparent blends with increased service temperature. With proper formulating, the higher HDT can be achieved without sacrificing other properties. It is especially important in clear PVC applications where no increase in haze was noted with the addition of the acrylic imide copolymer. Indications are that substantial market opportunities exist for a high heat PVC. Major market segments include clear packaging with it's need for hot-fill containers, building products where the desire to use darker colors provides the driving force, and in custom injection molding applications where improved heat resistance would allow vinyl to compete more effectively with FR-styrenics.     

Molders' perceptions of vinyl injection molding compounds

As a result of advances in such properties as processability and thermal stability, vinyl may now compete with ABS and many engineering resins in a broad number of injection molding applications. However, do past experiences with PVC compounds prevent molders from quoting on new vinyl jobs? What do molders think of vinyl's physical properties and molding characteristics versus other resins? Independent research conducted in 1989 questioned a broad range of molders in the United States regarding their perception of vinyl versus other resins as an injection molding material. The results highlight the fact that molders are aware of the benefits of vinyl in finished part applications, but require training to overcome the perceptions of past molding experiences. This paper shares the results of this survey.     

Neutrophil apoptosis is associated with a reduction in CD16 (Fc gamma RIII) expression

Resolution of inflammation involves removal of recruited neutrophils from inflamed sites via a noninflammatory mechanism, possibly involving neutrophil apoptosis and engulfment/phagocytosis by macrophages. In this study, we describe the reduction in surface expression (> 90%) of the neutrophil molecule Fc gamma RIII (CD16) during in vitro culture at 37 degrees C, which was found to be temporally associated with the appearance of neutrophils with apoptotic morphology during in vitro culture and inhibitable by granulocyte-macrophage colony-stimulating factor (GM-CSF), which postpones apoptosis in the neutrophil. By using dual fluorescence analysis, CD16 "low" expressing neutrophils showed reduced staining with the DNA-binding dye propidium iodide, suggesting that CD16 low expressing neutrophils were apoptotic. Separation of CD16 "high" and CD16 "low" expressing neutrophils by fluorescence-activated cell sorting revealed that morphologically apoptotic cells exhibited the CD16 low phenotype. We did not observe similar marked changes in expression of other neutrophil surface molecules (including other phosphatidylinositol (PI)-linked molecules), indicating that generalized loss of surface molecules does not occur during apoptosis. We believe this to be the first reported cell type-specific membrane alteration in a surface glycoprotein associated with apoptosis, suggesting that the program of cell death in the neutrophil, in addition to morphologic and nuclear changes, includes alterations in expression of surface receptors.     

Collaborative writing in multiple discourse contexts

Research in computer-supported writing has traditionally compared electronic communication with oral, face-to-face communication to identify the benefits and weaknesses of each, as if they entailed dichotomous choices. In this article, we challenge that view and argue instead that any form of communication and its educational usefulness is shaped by the situation in which it is used, the backgrounds and goals of the participants, the institutional and technological setup, and the intended purpose of the medium. Three modes of communication in one graduate course are examined — oral discussion, synchronous written discussion on a local area network, and asynchronous written postings on an email list set up for the class. It was found that patterns of participation, topic introduction, and topic development differed across the three communication modes, but that the three were interwoven with each other and embedded within the larger classroom context and forms of knowledge creation in the class. Thus, rather than examining different communication media separately, researchers interested in understanding computer-supported collaborative writing need to look at how different media are used to create a meta-medium, which is established by the discourse community involved.     

High-resolution crystal structures of human hemoglobin with mutations at tryptophan 37beta: structural basis for a high-affinity T-state,

The high-resolution X-ray structures of the deoxy forms of four recombinant hemoglobins in which Trp37(C3)beta is replaced with Tyr (betaW37Y), Ala (betaW37A), Glu (betaW37E), or Gly (betaW37G) have been refined and analyzed with superposition methods that partition mutation-induced perturbations into quaternary structure changes and tertiary structure changes. In addition, a new cross-validation statistic that is sensitive to local changes in structure (a "local Rfree" parameter) was used as an objective measure of the significance of the tertiary structure changes. No significant mutation-induced changes in tertiary structure are detected at the mutation site itself for any of the four mutants studied. Instead, disruption of the intersubunit contacts associated with Trp37(C3)beta results in (1) a change in quaternary structure at the alpha1beta2 interface, (2) alpha subunit tertiary structure changes that are centered at Asp94(G1)alpha-Pro95(G2)alpha, (3) beta subunit tertiary structure changes that are located between residues Asp99(G1)beta and Asn102(G4)beta, (4) increased mobility of the alpha subunit COOH-terminal dipeptide, and (5) shortening of the Fe-Nepsilon2His(F8) bond in the alpha and beta subunits of the betaW37G and betaW37E mutants. In each case, the magnitude of the change in a particular structural parameter increases in the order betaW37Y < betaW37A < betaW37E approximately betaW37G, which corresponds closely to the degree of functional disruption documented in the preceding papers.