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At least 901 records · Page 50Linked to original sources
PubMed2003

In search of the pathways for light-induced pacemaker resetting in the suprachiasmatic nucleus.

Within the suprachiasmatic nucleus (SCN) of the mammalian hypothalamus is a circadian pacemaker that functions as a clock. Its endogenous period is adjusted to the external 24-h light-dark cycle, primarily by light-induced phase shifts that reset the pacemaker's oscillation. Evidence using a wide variety of neurobiological and molecular genetic tools has elucidated key elements that comprise the visual input pathway for SCN photoentrainment in rodents. Important questions remain regarding the intracellular signals that reset the autoregulatory molecular loop within photoresponsive cells in the SCN's retino-recipient subdivision, as well as the intercellular coupling mechanisms that enable SCN tissue to generate phase shifts of overt behavioral and physiological circadian rhythms such as locomotion and SCN neuronal firing rate. Multiple neurotransmitters, protein kinases, and photoinducible genes add to system complexity, and we still do not fully understand how dawn and dusk light pulses ultimately produce bidirectional, advancing and delaying phase shifts for pacemaker entrainment.

Animals↗
PubMed2006

The methamphetamine-sensitive circadian oscillator (MASCO) in mice.

The suprachiasmatic nucleus (SCN) orchestrates synchrony among many peripheral oscillators and is required for circadian rhythms of locomotor activity and many physiological processes. However, the unique effects of methamphetamine (MAP) on circadian behavior suggest the presence of an SCN-independent, methamphetamine-sensitive circadian oscillator (MASCO). Substantial data collected using rat models show that chronic methamphetamine dramatically lengthens circadian period of locomotor activity rhythms and induces rhythms in animals lacking an SCN. However, the anatomical substrate and the molecular components of the MASCO are unknown. The response to MAP is less well studied in mice, a model that would provide the genetic tools to probe the molecular components of this extra-SCN oscillator. The authors tested the effects of chronic MAP on 2 strains of intact and SCN-lesioned mice in constant dark and constant light. Furthermore, they applied various MAP availability schedules to SCN-lesioned mice to confirm the circadian nature of the underlying oscillator. The results indicate that this oscillator has circadian properties. In intact mice, the MASCO interacts with the SCN in a manner that is strain, sex, and dose dependent. In SCN-lesioned mice, it induces robust free-running locomotor rhythmicity, which persists for up to 14 cycles after methamphetamine is withdrawn. In the future, localization of the MASCO and characterization of its underlying molecular mechanism, as well as its interactions with other oscillators in the body, will be essential to a complete understanding of the organization of the mammalian circadian system.

Animals↗
PubMed2006

Genetic considerations in thyroid cancer.

BACKGROUND: Recent molecular studies have described a number of abnormalities associated with the progression and dedifferentiation of thyroid carcinoma. These distinct molecular events are often associated with specific stages of tumor development. A better understanding of the mechanisms involved in thyroid cancer pathogenesis may help to translate these discoveries toward improvements in patient care. METHODS: We reviewed the literature on the molecular pathogenesis of thyroid cancer and compared clinical, histopathologic, and genetic features important in defining the disease process. RESULTS: The progression of thyroid cancer from well-differentiated to poorly differentiated and undifferentiated carcinoma represents a biological continuum. Specific genetic events serve as early initiating and late triggering events. Poorly differentiated thyroid carcinomas occupy an intermediate position in this progression model. CONCLUSIONS: With sophisticated genetic tools generating a wealth of information, we have gained better insight into the mechanisms driving thyroid tumor progression. Recognition of these features is crucial to the management of patients with thyroid cancer. Novel treatments are being designed based on our enhanced understanding of this disease process.

Adenocarcinoma, Follicular↗
PubMed2006

Comprehensive quality control utilizing the prehybridization third-dye image leads to accurate gene expression measurements by cDNA microarrays.

BACKGROUND: Gene expression profiling using microarrays has become an important genetic tool. Spotted arrays prepared in academic labs have the advantage of low cost and high design and content flexibility, but are often limited by their susceptibility to quality control (QC) issues. Previously, we have reported a novel 3-color microarray technology that enabled array fabrication QC. In this report we further investigated its advantage in spot-level data QC. RESULTS: We found that inadequate amount of bound probes available for hybridization led to significant, gene-specific compression in ratio measurements, increased data variability, and printing pin dependent heterogeneities. The impact of such problems can be captured through the definition of quality scores, and efficiently controlled through quality-dependent filtering and normalization. We compared gene expression measurements derived using our data processing pipeline with the known input ratios of spiked in control clones, and with the measurements by quantitative real time RT-PCR. In each case, highly linear relationships (R2 > 0.94) were observed, with modest compression in the microarray measurements (correction factor < 1.17). CONCLUSION: Our microarray analytical and technical advancements enabled a better dissection of the sources of data variability and hence a more efficient QC. With that highly accurate gene expression measurements can be achieved using the cDNA microarray technology.

Animals↗
PubMed2006

Functional characterization of a three-component regulatory system involved in quorum sensing-based regulation of peptide antibiotic production in Carnobacterium maltaromaticum.

BACKGROUND: Quorum sensing is a form of cell-to-cell communication that allows bacteria to control a wide range of physiological processes in a population density-dependent manner. Production of peptide antibiotics is one of the processes regulated by quorum sensing in several species of Gram-positive bacteria, including strains of Carnobacterium maltaromaticum. This bacterium and its peptide antibiotics are of interest due to their potential applications in food preservation. The molecular bases of the quorum sensing phenomenon controlling peptide antibiotic production in C. maltaromaticum remain poorly understood. The present study was aimed at gaining a deeper insight into the molecular mechanism involved in quorum sensing-mediated regulation of peptide antibiotic (bacteriocin) production by C. maltaromaticum. We report the functional analyses of the CS (autoinducer)-CbnK (histidine protein kinase)-CbnR (response regulator) three-component regulatory system and the three regulated promoters involved in peptide antibiotic production in C. maltaromaticum LV17B. RESULTS: CS-CbnK-CbnR system-dependent activation of carnobacterial promoters was demonstrated in both homologous and heterologous hosts using a two-plasmid system with a beta-glucuronidase (GusA) reporter read-out. The results of our analyses support a model in which the CbnK-CbnR two-component signal transduction system is necessary and sufficient to transduce the signal of the peptide autoinducer CS into the activation of the promoters that drive the expression of the genes required for production of the carnobacterial peptide antibiotics and the immunity proteins that protect the producer bacterium. CONCLUSIONS: The CS-CbnK-CbnR triad forms a three-component regulatory system by which production of peptide antibiotics by C. maltaromaticum LV17B is controlled in a population density-dependent (or cell proximity-dependent) manner. This regulatory mechanism would permit the bacterial population to synchronize the production of peptide antibiotics and immunity proteins. Such a population-wide action would afford a substantial peptide antibiotic production burst that could increase the ability of the bacterium to inhibit susceptible bacterial competitors. Finally, our CS-CbnK-CbnR-based two-plasmid expression system represents a suitable genetic tool for undertaking structure-function relationship analyses to map the amino acid residues in the components of the CS-CbnK-CbnR system that are required for biological activity. This plasmid system also has potential as a starting point for developing alternative vectors for controlled gene expression in C. maltaromaticum, Lactococcus lactis, and related lactic acid bacteria.

Anti-Bacterial Agents↗
PubMed2005

Single nucleotide polymorphisms (SNPs) are inherited from parents and they measure heritable events.

Single nucleotide polymorphisms (SNPs) are extensively used in case-control studies of practically all cancer types. They are used for the identification of inherited cancer susceptibility genes and those that may interact with environmental factors. However, being genetic markers, they are applicable only on heritable conditions, which is often a neglected fact. Based on the data in the nationwide Swedish Family-Cancer Database, we review familial risks for all main cancers and discuss the evidence for a heritable component in cancer. The available evidence is not conclusive but it is consistent in pointing to a minor heritable etiology in cancer, which will hamper the success of SNP-based association studies. Empirical familial risks should be used as guidance for the planning of SNP studies. We provide calculations for the assessment of familial risks for assumed allele frequencies and gene effects (odds ratios) for different modes of inheritance. Based on these data, we discuss the gene effects that could account for the unexplained proportion of familial breast and lung cancer. As a conclusion, we are concerned about the indiscriminate use of a genetic tool to cancers, which are mainly environmental in origin. We consider the likelihood of a successful application of SNPs in gene-environment studies small, unless established environmental risk factors are tested on proven candidate genes.

Editorial↗
PubMed1994

Molecular structure and regulation of vertebrate Na+/H+ exchangers.

Na+/H+ exchangers (NHE), also called antiporters, are vital transmembrane transporters involved in multiple cellular functions including the regulation of intracellular pH, the control of cell volume and transepithelial ion transport. These transporters are highly regulated by a remarkably wide variety of stimuli which can modulate their expression level and activity. Five isoforms of Na+/H+ exchangers have been cloned and characterized to date; they define a new gene family of vertebrate transporters. These isoforms share the same overall structure but exhibit differences with respect to amiloride-sensitivity, cellular localization, kinetic variables, regulation by various stimuli and plasma membrane targeting in polarized epithelial cells. Biochemical techniques and molecular genetics tools provide the means of analyzing these transporters at the molecular level. The purpose of this manuscript is to give an overview of the main features of the Na+/H+ exchangers with emphasis on recent advances in comprehension of the structure-function relationship and regulation mechanisms of the ubiquitous isoform: NHE-1.

Animals↗
PubMed2001

Skin reflectance in the Han Chinese and Tibetan populations.

Genetic and environmental factors are involved in the determination of skin pigmentation in humans. With the recent development of statistical and genetic tools in mapping complex traits in humans, it is becoming feasible to utilize such methods in identifying genes involved in skin pigmentation. Furthermore, the use of new portable reflectance spectroscopy instruments such as the Photovolt ColorWalk colorimeter allows researchers to measure skin reflectance of a large number of subjects with ease and accuracy. We used a new portable instrument (Photovolt ColorWalk) to study the skin reflectance of 372 Han Chinese and 274 Tibetan individuals to establish background reflectance measurements of unexposed skin of the inner upper arm in these two populations. In addition, we explored the effect of various factors such as age and gender on skin reflectance.

Adolescent↗
PubMed2004

Use of siRNA to study the function of MDC1 in DNA damage responses.

Small interfering RNA (siRNA) technology has emerged as a powerful genetic tool to investigate gene function in mammalian cells. Here we use siRNA to study a mediator of DNA damage-checkpoint protein 1 (MDC1), previously known as Kiaa0170 or NFBD1, in DNA damage responses. We show that MDC1 siRNA specifically and efficiently down-regulates MDC1 expression, resulting in defective radiation-induced apoptosis in A549 cells. Transfection with siRNA-resistant MDC1 restores radiation-induced apoptosis. These findings suggest that siRNA can be a very useful tool for the exploration of gene function in mammalian checkpoint responses.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Tetracycline-regulated gene expression in epidermal keratinocytes.

The tetracycline-regulated expression system developed by Gossen and Bujard is a powerful genetic tool that permits the expression of any gene construct introduced into either cultured cells or transgenic animals to be precisely controlled. It involves two components, a regulatory component based on the prokaryotic tetracycline repressor (TetR) and a response plasmid that expresses the gene of interest under control of the tetracycline-response element. In this paper, we review the Tet system methodology, discuss the available vector systems, and describe how to prepare and characterize keratinocyte cell lines that express a gene under tetracycline control. The methodology involves the development of stable cell lines expressing the TetR protein (either tTA or rtTA, expressed as a fusion with the VP16 activation domain), and a second set of double-stable cell lines that contain both TetR and the response plasmid (tetracycline-response element-gene X) expressed under tetracycline control. As an example of this methodology, we discuss our recently developed keratinocyte cell lines that express human filaggrin in a tetracycline- regulated manner. This technique, now also available in retrovirus and adenovirus-based vectors, is applicable both to the study of genes that are toxic to cells and more generally to understand how genes regulate cell structure/function, growth, and differentiation.

Cells, Cultured↗
PubMed2005

Replication and encapsidation of papillomaviruses in Saccharomyces cerevisiae.

Improvements in methodologies to recapitulate and study particular biological functions of the papillomavirus life cycle have led to great advances in our knowledge of these viruses. Described in this chapter are techniques that allow low-copy and high-copy replication of full-length human papillomavirus (HPV) genomes, as well as assembly of virus-like particles, in Saccharomyces cerevisiae (yeast). This system has several distinct advantages that make it an attractive complement to the well-established raft-culturing system. First, yeast are inexpensive, rapid, and simple to culture in the lab. Second, they provide an ever-widening array of genetic tools to analyze HPV functions--most recently notable, the yeast open reading frame (ORF)-deletion library. Third, yeast provide a potentially high-efficiency means to produce large quantities of infectious virus in a short time frame. Fourth, assembly of HPV virus in yeast allows encapsidation of mutant genomes, since previous studies have shown that no viral ORF is required for replication of full-length HPV in yeast.

Capsid↗
PubMed2005

Gonadotropin gene targeting and biological implications.

Pituitary gonadotropins FSH and LH are heterodimeric glycoproteins consisting of a common alpha and a hormone-specific beta subunit that are non-covalently linked. These hormones orchestrate gonadal growth, differentiation, and function by regulating both steroid-ogenesis and gametogenesis. Advances in the past two decades in manipulating the mouse genome by site-specific mutagenesis have heralded a new dimension to our understanding of the biology of gonadotropins. Using these gene-targeting approaches, knockout mice lacking the hormone-specific gonadotropin subunits, and hence the functional dimeric hormones, have been generated. These individual gonadotropin-deficient mice are useful to delineate the distinct in vivo biological roles of FSH and LH. These mice also serve as valuable genetic tools to study the signaling mechanisms within the gonads and help a better understanding of some forms of human infertility.

Animals↗
PubMed2004

RNA interference technologies for understanding and treating neurodegenerative diseases.

RNA interference (RNAi) is an evolutionarily conserved process that silences gene expression through double-stranded RNA species in a sequence-specific manner. With the completion of genome sequencing in multiple organisms, RNAi provides an efficient reverse genetics tool to reveal gene functions on a genome-wide scale. Conditional/inducible RNAi offers a new way to analyze gene function at different developmental stages and to create a new generation of animal models of human diseases. The sequence-specificity of RNAi and the fact that it is a naturally occurring process in human make it an excellent therapeutic tool for a wide range of diseases. This article provides a brief review of the current understandings of the mechanism of RNAi and its application to the nervous system, with particular focus on its application to understand mechanisms of neurodegenerative diseases. The prospects of the application of RNAi in clinical setting to treat these devastating diseases will also be presented.

Animals↗
PubMed2004

Recent advances towards identification of new drug targets for Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a very successful pathogen that remains a leading infectious killer worldwide. The global situation has become precarious due to various factors such as the variable efficacy of the Bacille Calmette-Guerin (BCG) vaccine, drug resistance, delay in diagnosis, association with HIV, and other factors, creating a long-lasting reservoir of impending disease and infection. Surprisingly, no new drugs have been developed in the past 30 years. The release of the complete genome sequence of M. tuberculosis and the availability of advanced genetic tools have provided the powerful repertoire of potential drug targets that are now in hand and can be explored in a more rational and directional manner. In this review, the authors highlight some possible therapeutic targets in M. tuberculosis. The gene products involved in various processes, such as mycobacterial cell wall synthesis, ability to acquire or obtain essential nutrients, persistence, transcription regulation, energy metabolism and others, such as the PE-PGRS family and culture filtrate proteins, would be potential targets for the development of new drugs. Apart from these categories, the importance of signal transduction events in the virulence of mycobacteria is discussed in relation to their potential as therapeutic targets. The potential of all of these therapeutic targets should be investigated together with the potential of being able to synthesise future chemotherapeutic agents.

Animals↗
PubMed2005

What have we learned about gonadotropin function from gonadotropin subunit and receptor knockout mice?

A number of biochemical and physiological studies elucidated the roles of pituitary and placental glycoprotein hormones. Advances in the past two decades in manipulating the mouse genome by random or site-specific mutagenesis have heralded a new dimension to our understanding of the biology of gonadotropins. It is now possible to model many human reproductive disorders involving gonadotropins/gonadotropin-signaling in the mouse. Mutant mice selectively lacking either FSH or LH or their cognate receptors have been generated. The gonadotropin ligand and the corresponding receptor knockout mice mostly phenocopy each other. Analyses with these genetic models confirmed earlier physiological studies; in addition they also revealed novel roles for gonadotropins previously unrecognized. While FSH action seems dispensable for male but not female fertility, absence of LH causes infertility in both the sexes. While Sertoli cell number and germ cell carrying capacity of the Sertoli cells in compromised in FSH mutants, both somatic and germ cell lineages are affected in the LH mutants resulting in complete male infertility. FSH mutant females demonstrate a preantral stage block in folliculogenesis and FSH alone is not sufficient to promote full folliculogenesis in the absence of LH. Pre-ovulatory stage follicles do not form and most of the follicles undergo apoptosis in the absence of LH. Many extra-gonadal phenotypes have been described for the receptor knockout mice and whether these bear any resemblances to those in patients with similar inactivating mutations in the receptors for FSH and LH remains an open question. Thus the in vivo models will continue to have a significant impact in understanding gonadotropin physiology and pathophysiology and serve as novel genetic tools to study signaling mechanisms in the gonads.

Animals↗
PubMed2005

Drosophila poly(ADP-ribose) glycohydrolase mediates chromatin structure and SIR2-dependent silencing.

Protein ADP ribosylation catalyzed by cellular poly(ADP-ribose) polymerases (PARPs) and tankyrases modulates chromatin structure, telomere elongation, DNA repair, and the transcription of genes involved in stress resistance, hormone responses, and immunity. Using Drosophila genetic tools, we characterize the expression and function of poly(ADP-ribose) glycohydrolase (PARG), the primary enzyme responsible for degrading protein-bound ADP-ribose moieties. Strongly increasing or decreasing PARG levels mimics the effects of Parp mutation, supporting PARG's postulated roles in vivo both in removing ADP-ribose adducts and in facilitating multiple activity cycles by individual PARP molecules. PARP is largely absent from euchromatin in PARG mutants, but accumulates in large nuclear bodies that may be involved in protein recycling. Reducing the level of either PARG or the silencing protein SIR2 weakens copia transcriptional repression. In the absence of PARG, SIR2 is mislocalized and hypermodified. We propose that PARP and PARG promote chromatin silencing at least in part by regulating the localization and function of SIR2 and possibly other nuclear proteins.

Adenosine Diphosphate Ribose↗
PubMed2002

Rapid identification of mitochondrial cytochrome B haplotypes by single strand conformation polymorphism in Lutzomyia longipalpis (Diptera: Psychodidae) populations.

Newly designed primers amplified a 365-bp fragment including the 3' end of mitochondrial cytochromeb (cyt b). Field collected specimens of the sand fly, Lutzomyia longipalpis (Lutz and Neiva), from two Brazil locations provided test DNA. The primers, based on consensus sequences from a taxonomic spectrum of arthropods, have also amplified DNA successfully from Lutzomyia shannoni Dyar, Lutzomyia cruzi (Mangabeira), and the mosquito Aedes albopictus (Skuse). Single strand conformation polymorphism (SSCP) was used to recognize the presence of L. longipalpis haplotypes; these were verified subsequently by sequencing the polymerase chain reaction products. The sensitivity of SSCP was demonstrated by (1) the detection of single nucleotide differences in SSCP variants and (2) no sequence variation in specimens with the same SSCP mobility. The application of new primers and the SSCP technique provided a valuable addition to available population genetic tools: they increased the efficiency of detection of variability in the cyt b gene and decreased the time required for screening large numbers of specimens to detect nucleotide variation.

Animals↗
PubMed2002

Cytotoxic agents in the era of molecular targets and genomics.

Cancer treatment is evolving due to the development of molecularly targeted agents and the utilization of pharmacogenomics and pharmacogenetics to identify patients who are at an increased risk for toxicity or may be uniquely responsive to cytotoxic therapies. By identifying polymorphisms in the human genome that confer changes in the ability to metabolize or activate cancer agents, a more patient-specific treatment approach can be initiated. Molecularly targeted therapies such as PS-341, flavopiridol, Iressa, and anti-vascular endothelial growth factor antibodies may help to overcome resistance to cytotoxic therapies by lowering the apoptotic threshold and increasing cytotoxicity. Using molecularly targeted agents in combination with traditional cytotoxic agents may increase the percentage of patients who achieve disease stabilization and prolonged survival. With the development of genetic tools and genotyping of tumor and patient prior to initiating treatment, antitumor efficacy may be increased with a substantial reduction in toxicity.

Antineoplastic Agents, Alkylating↗