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PubMedDate not supplied

Biotechnological methods to accelerate cheddar cheese ripening.

Cheese is one of the dairy products that can result from the enzymatic coagulation of milk. The basic steps of the transformation of milk into cheese are coagulation, draining, and ripening. Ripening is the complex process required for the development of a cheese's flavor, texture and aroma. Proteolysis, lipolysis and glycolysis are the three main biochemical reactions that are responsible for the basic changes during the maturation period. As ripening is a relatively expensive process for the cheese industry, reducing maturation time without destroying the quality of the ripened cheese has economic and technological benefits. Elevated ripening temperatures, addition of enzymes, addition of cheese slurry, attenuated starters, adjunct cultures, genetically engineered starters and recombinant enzymes and microencapsulation of ripening enzymes are traditional and modern methods used to accelerate cheese ripening. In this context, an up to date review of Cheddar cheese ripening is presented.

Biotechnology↗
PubMedDate not supplied

Use of genetically engineered microorganisms (GEMs) for the bioremediation of contaminants.

This paper presents a critical review of the literature on the application of genetically engineered microorganisms (GEMs) in bioremediation. The important aspects of using GEMs in bioremediation, such as development of novel strains with desirable properties through pathway construction and the modification of enzyme specificity and affinity, are discussed in detail. Particular attention is given to the genetic engineering of bacteria using bacterial hemoglobin (VHb) for the treatment of aromatic organic compounds under hypoxic conditions. The application of VHb technology may advance treatment of contaminated sites, where oxygen availability limits the growth of aerobic bioremediating bacteria, as well as the functioning of oxygenases required for mineralization of many organic pollutants. Despite the many advantages of GEMs, there are still concerns that their introduction into polluted sites to enhance bioremediation may have adverse environmental effects, such as gene transfer. The extent of horizontal gene transfer from GEMs in the environment, compared to that of native organisms including benefits regarding bacterial bioremediation that may occur as a result of such transfer, is discussed. Recent advances in tracking methods and containment strategies for GEMs, including several biological systems that have been developed to detect the fate of GEMs in the environment, are also summarized in this review. Critical research questions pertaining to the development and implementation of GEMs for enhanced bioremediation have been identified and posed for possible future research.

Bacteria↗
PubMed2005

Biofilm formation by bacterial associations under various salinities and copper ion stress.

The study addresses the effect of abiotic (medium salinity and copper ions) and biotic (interactions between populations) factors on the formation of structured communities by binary associations consisting of halotolerant bacteria (Alcaligenes sp. 1-1 or Acinetobacter sp. 1-19) and a wild-type B. subtilis 2335 strain or a transgenic strain. The results showed that 250 mg l(-1) of copper ions inhibit formation of biofilms by monocultures of the tested strains. Binary associations of the strains were more resistant to high concentrations (250 mg l(-1)) of copper ions. At the lowest NaCl concentration (0.05% and 2.5%) and in the presence of copper ions, bacilli seemed to help halotolerant bacteria survive. Under increased salinity and in the presence of copper ions, structured communities developed due to halotolerant bacteria. Coexistence under stressful conditions was beneficial for the both groups of bacteria.

Acinetobacter↗
PubMed2005

Notes on "bioethics and sin" by Jean-Francois Collange.

Placing the notion of sin in the context of a meontic account of evil, and emphasizing the effect of sin on the sinner himself, this commentary exposes the insufficiency of restricting oneself to human efforts at atonement, and of thus underemphasizing the role of Christ. Collange's claim that the teaching of "predestination" is rooted in Paul and that the doctrine of merits and indulgences is rooted in Augustine is criticized, and Luther's "forensic" understanding is linked with Augustine, rather than with Paul. Collange's reduction of the concern for holiness to respect and trust is contrasted with holiness's essential context of loving unification with God. The commentary closes by exposing the unsatisfactory scantiness of Collange's treatment of cloning, health-care economy, and of the evils of life.

Bioethics↗
PubMed2003

Cdc48p is required for the cell cycle commitment point at Start via degradation of the G1-CDK inhibitor Far1p.

The budding yeast Cdc48p and its mammalian homologue p97 are involved in many important cellular activities. Because previous cdc48 mutants have exclusive G2/M arrest, Cdc48p was thought to play an essential role only during mitosis. We found that Cdc48p is required for the execution of Start (a yeast cell cycle commitment point equivalent to the restriction point in mammalian cells) in both a normal mitotic cell cycle and cell cycle reentry after mating pheromone withdrawal through degradation of the G1-cyclin-dependent kinase inhibitor Far1p. Our work is the first to uncover novel roles of Cdc48p as a critical cell cycle regulator in G1, and to shed new light on cell cycle regulation of Far1p, which is the first cyclin-dependent kinase inhibitor shown to be a substrate of an essential proteolysis event mediated by Cdc48p.

Adenosine Triphosphatases↗
PubMedDate not supplied

Experimental evolution and the Krogh principle: generating biological novelty for functional and genetic analyses.

August Krogh counseled the careful selection of the best subject organism on which to undertake mechanistic physiological research. But what if an organism with the desired properties does not exist? It is now within our power to engineer organisms genetically to achieve novel combinations of traits. I propose that it is a logical extension of the Krogh principle that we use biological methodologies to create novel organisms ideally suited for particular physiological studies. Transgenics may first come to mind as the method for such transformations, but here I suggest that an alternative and complementary technique for generating biological novelty is experimental evolution. The latter has several advantages, including modification of multiple characters in one experiment, the production of advantageous traits, the testing of evolutionary hypotheses, and the identification of previously unsuspected factors involved in adaptation. Three experiments are reviewed, each of which examined the evolution of different physiological characters in different environments and organisms: locomotor performance in mice, desiccation tolerance in fruit flies, and high temperature adaptation in bacteria. While diverse in experimental type and subject, all resulted in the successful production of new variants with enhanced function in their new environments. Each experiment successfully tested hypotheses concerning physiological evolution, and in each case, unanticipated results emerged, which suggests previously unsuspected adaptive pathways and mechanisms. In addition, replicate populations in each experiment adjusted to their common environments by several different means, which indicates that physiological evolution may follow diverse stochastic pathways during adaptation. Experimental evolution can be a valuable method to produce and investigate new physiological variants and traits. The choice of experimental subjects, according to the Krogh principle, is no longer limited to currently existing organisms but is open to our imaginations and our ingenuity.

Adaptation, Physiological↗
PubMed2006

Baculovirus surface display of SARS coronavirus (SARS-CoV) spike protein and immunogenicity of the displayed protein in mice models.

The baculovirus surface display technique has provided an ideal tool to display foreign proteins with natural conformation, functions, and immunogenicity. In this work, we explored the application of this technique on SARS-associated coronavirus (SARS-CoV) spike (S) protein, and further analyzed the immunogenicity of displayed S protein. The entire ectodomain of S protein was fused between the gp64 signal peptide and the VSV-G membrane anchor and successfully displayed on the baculovirus surface. Subcutaneous injection with purified S-displayed baculoviruses without adjuvant elicited highly effective production of specific and neutralizing antibodies against S protein in mice. These results confirmed a successful surface display of S protein on baculoviruse, and suggested a potential role of S-displayed baculoviruses as a novel live virus-based vaccine candidate for SARS-CoV.

Animals↗
PubMed2005

Yeast aconitase in two locations and two metabolic pathways: seeing small amounts is believing.

The distribution of identical enzymatic activities between different subcellular compartments is a fundamental process of living cells. At present, the Saccharomyces cerevisiae aconitase enzyme has been detected only in mitochondria, where it functions in the tricarboxylic acid (TCA) cycle and is considered a mitochondrial matrix marker. We developed two strategies for physical and functional detection of aconitase in the yeast cytosol: 1) we fused the alpha peptide of the beta-galactosidase enzyme to aconitase and observed alpha complementation in the cytosol; and 2) we created an ACO1-URA3 hybrid gene, which allowed isolation of strains in which the hybrid protein is exclusively targeted to mitochondria. These strains display a specific phenotype consistent with glyoxylate shunt elimination. Together, our data indicate that yeast aconitase isoenzymes distribute between two distinct subcellular compartments and participate in two separate metabolic pathways; the glyoxylate shunt in the cytosol and the TCA cycle in mitochondria. We maintain that such dual distribution phenomena have a wider occurrence than recorded currently, the reason being that in certain cases there is a small fraction of one of the isoenzymes, in one of the locations, making its detection very difficult. We term this phenomenon of highly uneven isoenzyme distribution "eclipsed distribution."

Acetates↗
PubMed2005

Functions of the small proteins in the TOM complex of Neurospora crasssa.

The TOM (translocase of the outer mitochondrial membrane) complex of the outer mitochondrial membrane is required for the import of proteins into the organelle. The core TOM complex contains five proteins, including three small components Tom7, Tom6, and Tom5. We have created single and double mutants of all combinations of the three small Tom proteins of Neurospora crassa. Analysis of the mutants revealed that Tom6 plays a major role in TOM complex stability, whereas Tom7 has a lesser role. Mutants lacking both Tom6 and Tom7 have an extremely labile TOM complex and are the only class of mutant to exhibit an altered growth phenotype. Although single mutants lacking N. crassa Tom5 have no apparent TOM complex abnormalities, studies of double mutants lacking Tom5 suggest that it also has a minor role in maintaining TOM complex stability. Our inability to isolate triple mutants supports the idea that the three proteins have overlapping functions. Mitochondria lacking either Tom6 or Tom7 are differentially affected in their ability to import different precursor proteins into the organelle, suggesting that they may play roles in the sorting of proteins to different mitochondrial subcompartments. Newly imported Tom40 was readily assembled into the TOM complex in mitochondria lacking any of the small Tom proteins.

Fungal Proteins↗
PubMed2002

Distinct mechanisms of E2F regulation by Drosophila RBF1 and RBF2.

RBF1, a Drosophila pRB family homolog, is required for cell cycle arrest and the regulation of E2F-dependent transcription. Here, we describe the properties of RBF2, a second family member. RBF2 represses E2F transcription and is present at E2F-regulated promoters. Analysis of in vivo protein complexes reveals that RBF1 and RBF2 interact with different subsets of E2F proteins. dE2F1, a potent transcriptional activator, is regulated specifically by RBF1. In contrast, RBF2 binds exclusively to dE2F2, a form of E2F that functions as a transcriptional repressor. We find that RBF2-mediated repression requires dE2F2. More over, RBF2 and dE2F2 act synergistically to antagonize dE2F1-mediated activation, and they co-operate to block S phase progression in transgenic animals. The network of interactions between RBF1 or RBF2 and dE2F1 or dE2F2 reveals how the activities of these proteins are integrated. These results suggest that there is a remarkable degree of symmetry in the arrangement of E2F and RB family members in mammalian cells and in DROSOPHILA.

Amino Acid Sequence↗
PubMed2003

Regulation of light-dependent Gqalpha translocation and morphological changes in fly photoreceptors.

Heterotrimeric G-proteins relay signals between membrane-bound receptors and downstream effectors. Little is known, however, about the regulation of Galpha subunit localization within the natural endogenous environment of a specialized signaling cell. Here we show, using live Drosophila flies, that light causes massive and reversible translocation of the visual Gqalpha to the cytosol, associated with marked architectural changes in the signaling compartment. Molecular genetic dissection together with detailed kinetic analysis enabled us to characterize the translocation cycle and to unravel how signaling molecules that interact with Gqalpha affect these processes. Epistatic analysis showed that Gqalpha is necessary but not sufficient to bring about the morphological changes in the signaling organelle. Furthermore, mutant analysis indicated that Gqbeta is essential for targeting of Gqalpha to the membrane and suggested that Gqbeta is also needed for efficient activation of Gqalpha by rhodopsin. Our results support the 'two-signal model' hypothesis for membrane targeting in a living organism and characterize the regulation of both the activity-dependent Gq localization and the cellular architectural changes in Drosophila photoreceptors.

Actins↗
PubMed2003

A test of insulator interactions in Drosophila.

Insulators are a class of elements that define independent domains of gene function. The Drosophila gypsy insulator is proposed to establish regulatory isolation by forming loop domains that constrain interactions between transcriptional control elements. This supposition is based upon the observation that insertion of a single gypsy insulator between an enhancer and promoter blocks enhancer function, while insertion of two gypsy insulators promotes enhancer bypass and activation of transcription. To investigate this model, we determined whether non-gypsy insulators interacted with each other and with the gypsy insulator. Pairs of scs or scs' insulators blocked enhancer function. Further, an intervening scs insulator did not block gypsy insulator interactions. Taken together, these data suggest that not all Drosophila insulators interact, with this property restricted to some insulators, such as gypsy. Three gypsy insulators inserted between an enhancer and promoter blocked enhancer function, indicating that gypsy insulator interactions may be restricted to pairs. Our studies imply that formation of loop domains may represent one of many mechanisms used by insulators to impart regulatory isolation.

Animals↗