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At least 595 records · Page 33Linked to original sources
PubMed2002

A nucleotide substitution in the tRNA(Lys) primer binding site dramatically increases replication of recombinant simian immunodeficiency virus containing a human immunodeficiency virus type 1 reverse transcriptase.

A recombinant simian immunodeficiency virus (SIV) derived from strain 239 (SIVmac239) with reverse transcriptase (RT) sequences from human immunodeficiency virus type 1 (HIV-1) strain HXB2 was severely impaired for replication. Detectable p27(Gag) levels were not observed until day 65 and peak p27(Gag) levels were not reached until day 75 after transfection of CEMx174 cells with the recombinant DNA. Sequences from the latter time point did not contain amino acid substitutions in HIV-1 RT; however, a single nucleotide substitution (thymine to cytosine) was found at position eight of the SIV primer binding site. We engineered an RT/SHIV genome with the thymine-to-cytosine substitution, called RT/SHIV/TC, and observed dramatically faster replication kinetics than were observed with the parental RT/SHIV from which this variant was derived. RT/SHIV/TC provides an improved system for study of the impact of drug resistance mutations in HIV-1 RT in a relevant animal model.

Amino Acid Substitution↗
PubMed2001

PROBING PLANT METABOLISM WITH NMR.

Analytical methods for probing plant metabolism are taking on new significance in the era of functional genomics and metabolic engineering. Among the available methods, nuclear magnetic resonance (NMR) spectroscopy is a technique that can provide insights into the integration and regulation of plant metabolism through a combination of in vivo and in vitro measurements. Thus NMR can be used to identify, quantify, and localize metabolites, to define the intracellular environment, and to explore pathways and their operation. We review these applications and their significance from a metabolic perspective. Topics of current interest include applications of NMR to metabolic flux analysis, metabolite profiling, and metabolite imaging. These and other areas are discussed in relation to NMR investigations of intermediary carbon and nitrogen metabolism. We conclude that metabolic NMR has a continuing role to play in the development of a quantitative understanding of plant metabolism and in the characterization of metabolic phenotypes.

Journal Article↗
PubMed2002

Of mice and models: improved animal models for biomedical research.

The ability to engineer the mouse genome has profoundly transformed biomedical research. During the last decade, conventional transgenic and gene knockout technologies have become invaluable experimental tools for modeling genetic disorders, assigning functions to genes, evaluating drugs and toxins, and by and large helping to answer fundamental questions in basic and applied research. In addition, the growing demand for more sophisticated murine models has also become increasingly evident. Good state-of-principle knowledge about the enormous potential of second-generation conditional mouse technology will be beneficial for any researcher interested in using these experimental tools. In this review we will focus on practice, pivotal principles, and progress in the rapidly expanding area of conditional mouse technology. The review will also present an internet compilation of available tetracycline-inducible mouse models as tools for biomedical research (http://www.zmg.uni-mainz.de/tetmouse/).

Animals↗
PubMed2004

Information management for entomology screening.

The successful introduction of genetically modified (GM) crops has created a revolutionary transformation of the agricultural industry with GM crops currently planted on some 130 million acres. Historically, agricultural companies were involved in the discovery of conventional insecticide chemistries in processes that were very similar to those in the pharmaceutical industry. With the introduction of GM technologies, the process has radically changed. The new process is best described as the pharmaceutical equivalent of a very large-scale limited clinical trial conducted at the discovery stage. For example, unlike pharmaceutical screens, GM crop discovery screens frequently involve live insects challenged with complex protein containing soups. Furthermore, the nature of the potential avenues for discovery mandates that assay models also support protein engineering, proteomics, and genomics efforts. The range in sample demand ranges from ultra-low to high throughput. Finally, regulatory and good business stewardship demands that data integrity and tractability is in place, from discovery through varietal introduction, to manage the information generated in support of a commercialized GM plant variety. The authors describe their discovery workflow and detail how they have customized a commercially available database software package to achieve an optimum configuration for entomology screening.

Animals↗
PubMed2004

Gene delivery to the mouse brain with adeno-associated virus.

The efficient transduction of postmitotic cells by adeno-associated virus (AAV) makes it an excellent vector to deliver marker, functional, or therapeutic genes to the mammalian brain. An attractive feature of AAV is that all the viral-coding sequences are removed when engineering the recombinant genome, thereby limiting the extent of cell toxicity and immune response that are often associated with viral gene transcription. Of the seven described AAV serotypes, AAV serotype-2 (AAV2) is the most studied gene-transfer vehicle for in the mammalian brain. A feature of AAV2 transduction in the brain is that the vector remains confined to the injection site and predominately infects neurons rather than glia (2-8). The limited diffusion of AAV2 vectors is beneficial for controlled gene delivery. For instance, targeting therapeutic genes only to brain structures showing pathology would eliminate complications associated with vector diffusion and subsequent expression in healthy structures, and is an important consideration when designing treatment strategies for localized neurodegenerative diseases. The same is true for other experimental paradigms, such as investigating the function of genes in specific brain structures or using marker genes in tract-tracing experiments. Although AAV2 vectors were shown to remain predominately at the injection site, one study demonstrated that the vector itself may undergo axonal transport in inter-regional systems (9).

Animals↗
PubMed2005

Gene targeting and transgenic strategies for the analysis of hematopoietic development in the mouse.

The generation of gene-targeted and transgenic mouse models facilitates the in vivo study of mammalian gene function. Advances in technologies to engineer the mouse genome have extended the choice of gene manipulation from straightforward gene inactivation or overexpression to detailed modification of gene expression pattern, structure, and function in desired cell types and at specific times. Combining conventional/conditional, knockout/knockin, inducible, and even reversible gene manipulation strategies provides the investigator with the freedom to design an optimal model to study the function of a gene in a specific organ system during development or in postnatal life. To maximize success, however, the requirements and limitations of each approach need to be considered. This chapter provides an overview of gene targeting strategies that are available for manipulation of the mouse genome. We emphasize approaches that aid the investigation of the development and function of the hematopoietic system in the mouse.

Animals↗
PubMed2002

Cancer-homing toxins.

Cancer-homing toxins are a group of man-made cytotoxic molecules targeting cancer cells. In the past decade they have demonstrated potential as cancer therapeutics. These molecules contain a toxin, natural or usually derivatized, connected to a cancer-homing module, such as a monoclonal antibody or growth factor or their derivatives. Various cancer-homing toxins have been designed and tested in cell-lines, animal-models and clinical trials. We review some of these data and discuss ways to better design cancer-homing toxins in the light of advances in cancer genomics, antibody-engineering techniques and computational algorithms.

Antibodies, Monoclonal↗
PubMed1976

Restriction endonucleases.

This review provides a comprehensive account of the current status of the biology and biochemistry of restriction endonucleases. Both Class I and Class II restriction endonucleases will be considered. However, emphasis will be placed on the Class II group, which recognizes and cleaves a specific duplex DNA sequence. Their occurrence, purification, and characterization is discussed in detail. The characterization includes physical mapping information and determination of recognition sequences. In addition to detailed discussions of the biochemical properties of the enzymes, considerable attention is paid to the uses of these enzymes as tools for research in molecular biology. These uses include physical mapping of genomes and their transcripts, genetic analysis (marker rescue, etc.), DNA sequence analysis, analysis of complex genomes, and genetic engineering. Specific examples of each use are outlined. Practical aspects of both the isolation and use of the restriction endonucleases form the major theme of this review.

Bacteria↗
PubMed2026

Hormone priming and metabolic engineering of phytohormone crosstalk in rice under combined biotic and abiotic stresses: a multi-omics perspective for climate-resilient crop development.

Rice (Oryza sativa L.) is the caloric backbone for more than half of humanity, yet it remains one of the most vulnerable crops to the simultaneous biotic and abiotic stresses exacerbated by climate change. Phytohormone priming and the complex crosstalk networks governed by transcription factor hubs like WRKY, MYB, and NAC serve as the central adaptive mechanism for stress resilience. This review synthesizes how multi-omics integration, including spatial and single-cell transcriptomics, is resolving the molecular architecture of hormonal priming and epigenetic stress memory. We critically evaluate advanced metabolic engineering and genome-editing strategies such as CRISPR-Cas9, base/prime editing, and synthetic gene circuits that enable precision modifications to decouple stress tolerance from historical yield penalties. Furthermore, we discuss the emerging roles of microbiome-assisted priming via synthetic consortia and the application of artificial intelligence and digital twins (continuously updated computational models of crop physiology) for predictive stress management. By integrating these diverse technological pillars, we propose a systems-level roadmap for developing climate-resilient rice cultivars capable of maintaining yield stability across a volatile combinatorial stress landscape. This synthesis provides a framework for translating mechanistic hormonal insights into field-applicable cultivars to ensure global food security.

CRISPR↗
PubMed2026

Brassinosteroids as Central Regulators of Plant Growth, Stress Tolerance, and Agricultural Resilience.

Brassinosteroids (BRs) are essential steroidal phytohormones that regulate plant growth, development, and responses to environmental stresses. Recent studies have demonstrated the important roles of BRs in enhancing plant tolerance to abiotic stresses, including drought, salinity, temperature extremes, heavy metal toxicity, and oxidative stress, as well as biotic stresses caused by pathogens and herbivores. This review summarizes current advances in BR biosynthesis, metabolism, transport, and signaling pathways, focusing on key components that mediate stress adaptation. We discuss the physiological and molecular mechanisms through which BRs improve stress tolerance, including regulation of antioxidant defense, ion homeostasis, osmotic adjustment, and stress-responsive gene expression. Particular attention is given to the extensive cross talk between BRs and other phytohormones, such as abscisic acid, jasmonic acid, salicylic acid, ethylene, auxin, and gibberellins, which enables plants to balance growth and defense under adverse conditions. Furthermore, we highlighted the potential applications of BRs in crop improvement through exogenous treatments, genetic engineering, and genome-editing approaches. However, the effectiveness of BR-based strategies is highly dependent on crop species, developmental stage, stress type, BR concentration, application method, and environmental conditions. In addition, excessive BR accumulation or application may result in undesirable growth responses, and further multi-location field validation is required before widespread agricultural implementation. Finally, we discuss emerging research trends, current knowledge gaps, and future perspectives for exploring BR signaling to develop climate-resilient crops. Overall, BRs represent promising targets for improving crop stress resilience; however, optimizing BR-mediated strategies and validating their long-term performance under diverse field conditions will be essential for their successful application in sustainable agriculture.

abiotic stress↗
PubMed2006

Construction of an oral recombinant DNA vaccine from H pylori neutrophil activating protein and its immunogenicity.

AIM: To construct a live attenuated Salmonella typhimurium (S. typhimurium) strain harboring the H pylori neutrophil activating protein (HP-NAP) gene as an oral recombinant DNA vaccine, and to evaluate its immunogenicity. METHODS: By genetic engineering methods, the genomic DNA of H pylori was extracted as a template. The total length of the HP-NAP gene was amplified by polymerase chain reaction (PCR) and cloned into pBT vector for sequencing and BLAST analysis, then subcloned into a eukaryotic expression vector pIRES followed by PCR identification and restriction enzyme digestion. The identified recombinant plasmid pIRES-NAP was transfected into COS-7 cells for target fusion protein expression, and its antigenicity was detected by Western blotting. Then the recombinant plasmid was transformed into a live attenuated S. typhimurium strain SL7207 as an oral vaccine strain, and its immunogenicity was evaluated with animal experiments. RESULTS: A 435 bp product was cloned using high homology with HP-NAP gene in GenBank (more than 98%). With identification by PCR and restriction enzyme digestion, a recombinant eukaryotic expression plasmid pIRES-NAP containing the HP-NAP gene of H pylori was successfully constructed. The expressed target protein had a specific reaction with H pylorii whole cell antibody and showed a single strip result detected by Western blotting. Oral immunization of mice with recombinant DNA vaccine strain SL7207 (pIRES-NAP) also induced a specific immune response. CONCLUSION: The successful construction of HP-NAP oral DNA vaccine with good immunogenicity may help to further investigate its immunoprotection effects and develop vaccine against H pylori infection.

Administration, Oral↗
PubMed1998

Parallel Protein Information Analysis (PAPIA) System Running on a 64-Node PC Cluster.

Protein information analysis is widely regarded as a key technology in drug design, macromolecular engineering, and understanding genome sequences. Because vast amount of calculations are required, further speed-up for protein information analysis is very much in demand. We have implemented the PAPIA (PArallel Protein Information Analysis) system on the RWC PC cluster IIa (PAPIA cluster) which consists of 64 Pentium Pro 200MHz microprocessors. The PAPIA system performs fast parallel processing for typical calculations in protein analysis, such as structure similarity search, sequence homology search and multiple sequence alignment, nearly 60 times faster than a single processor. We have started a WWW service (http://www.rwcp.or.jp/papia/), allowing any biologist to easily submit jobs to the PAPIA system through a WWW browser. The user can experience the power of current parallel processing technology.

Journal Article↗
PubMed1992

Multipurpose vectors for peptide expression on the M13 viral surface.

We have developed a set of three cloning vectors for the expression of polypeptides on the surface of the M13 viral coat. The M13mp8 genome has been engineered for expression of foreign protein sequences near the NH2-terminus of the mature pIII protein, which is present in five copies on the outside of each M13 viral particle. All three of the vectors carry the same two useful restriction sites for directed cloning of inserts in the pIII coding region; in addition, one vector carries the bacterial gene conferring resistance to the antibiotic tetracycline, and another expresses the lacZ' polypeptide that allows functional complementation of beta-galactosidase activity within the host bacterial cell. All of these vectors propagate well in E. coli DH5 alpha F' cells and do not require helper phage. We demonstrate that a bacteriophage, expressing an eleven amino acid epitope (from human c-myc) at the NH2-terminus of pIII in one of our vectors, can be purified from a vast mixture of other M13 phage through panning techniques. In particular, we find that the c-myc-expressing viral particles can be easily recovered from phage mixtures with the biotinylated form of the monoclonal antibody, 9E10, and streptavidin-coated MagneSphere beads.

Amino Acid Sequence↗
PubMed2026

Genomic features, metabolism, and biotechnological applications of Candida tropicalis and other non-albicans Candida species.

The production of bio-based products by yeasts from agroindustrial byproducts is a key strategy for advancing circular bioeconomy. While Saccharomyces species remain the predominant industrial yeasts, their limited ability to assimilate lactose, pentoses, and glycerol, as well as their sensitivity to lignocellulose-derived inhibitors, restricts their efficient application in bioprocesses based on using industrial byproducts as fermentation media. In contrast, several non-albicans Candida species exhibit broad substrate utilization capacities and enhanced tolerance to industrial stresses, making them attractive candidates for the bioconversion of agroindustrial residues. This review critically examines recent advances in the genomic, metabolic, and physiological characterization of promising non-albicans Candida species, including Candida tropicalis, Candida parapsilosis, Candida viswanathii, Candida sojae, and Candida maltosa. Emphasis is given to genome-scale metabolic models, carbon assimilation pathways, stress-response mechanisms, and metabolic engineering approaches aiming at the production of value-added compounds. By identifying current achievements, knowledge gaps, and biotechnological bottlenecks, this review highlights the potential of these yeasts as emerging platforms for sustainable bioprocesses within a circular bioeconomy framework.

Biotechnology↗
PubMed2004

Genome-derived vaccines.

Vaccine research entered a new era when the complete genome of a pathogenic bacterium was published in 1995. Since then, more than 97 bacterial pathogens have been sequenced and at least 110 additional projects are now in progress. Genome sequencing has also dramatically accelerated: high-throughput facilities can draft the sequence of an entire microbe (two to four megabases) in 1 to 2 days. Vaccine developers are using microarrays, immunoinformatics, proteomics and high-throughput immunology assays to reduce the truly unmanageable volume of information available in genome databases to a manageable size. Vaccines composed by novel antigens discovered from genome mining are already in clinical trials. Within 5 years we can expect to see a novel class of vaccines composed by genome-predicted, assembled and engineered T- and Bcell epitopes. This article addresses the convergence of three forces--microbial genome sequencing, computational immunology and new vaccine technologies--that are shifting genome mining for vaccines onto the forefront of immunology research.

Animals↗
PubMed1991

Solid-phase gene assembly.

New technologies for multiple chemical synthesis of oligonucleotides and stepwise hybridization on a solid-phase support enable the rapid and cost-effective preparation of long duplex DNA regions. Will these new technologies usher in a new era in protein engineering?

DNA↗
PubMed1983

Molecular Engineering of the Autographa californica Nuclear Polyhedrosis Virus Genome: Deletion Mutations Within the Polyhedrin Gene.

We describe a method to introduce site-specific mutations into the genome of Autographa californica nuclear polyhedrosis virus. Specifically, the A. californica nuclear polyhedrosis virus gene for polyhedrin, the major protein that forms viral occlusions in infected cells, was mutagenized by introducing deletions into the cloned DNA fragment containing the gene. The mutagenized polyhedrin gene was transferred to the intact viral DNA by mixing fragment and viral DNAs, cotransfecting Spodoptera frugiperda cells, and screening for viral recombinants that had undergone allelic exchange. Recombinant viruses with mutant polyhedrin genes were obtained by selecting the progeny virus that did not produce viral occlusions in infected cells (occlusion-negative mutants). Analyses of occlusion-negative mutants demonstrated that the polyhedrin gene was not essential for the production of infectious virus and that deletion of certain sequences within the gene did not alter the control, or decrease the level of expression, of polyhedrin. An early viral protein of 25,000 molecular weight was apparently not essential for virus replication in vitro, as the synthesis of this protein was not detected in cells infected with a mutant virus.

Journal Article↗