Chronic antagonism of melanocortin receptors by the paracrine-acting agouti gene product induces both yellow fur and a maturity-onset obesity syndrome in mice that ubiquitously express wild-type agouti. Functional analysis of agouti mutations in transgenic mice indicate that the cysteine-rich C terminus, signal peptide, and glycosylation site are required for agouti activity in vivo. In contrast, no biological activity has been ascribed to the conserved basic domain. To examine the functional significance of the agouti basic domain, the entire 29-aa region was deleted from the agouti cDNA, and the resulting mutation (agoutiΔbasic) was expressed in transgenic mice under the control of the β-actin promoter (BAPaΔbasic). Three independent lines of BAPaΔbasic transgenic mice all developed some degree of yellow pigment in the fur, indicating that the agoutiΔbasic protein was functional in vivo. However, none of the BAPaΔbasic transgenic mice developed completely yellow fur, obesity, hyperinsulinemia, or hyperglycemia. High levels of agoutiΔbasic expression in relevant tissues exceeded the level of agouti expression in obese viable yellow mice, suggesting that suboptimal activity or synthesis of the agoutiΔbasic protein, rather than insufficient RNA synthesis...
The Hairy-related transcription-factor
(HRT) genes encode three related basic
helix–loop–helix transcription factors that show sequence similarity
to the Hairy and Enhancer of split family of transcriptional
repressors. HRT proteins are expressed in specific regions of the
developing heart, vasculature, pharyngeal arches and somites, and the
periodicity of their expression in somitic precursors mirrors that of
Notch signaling-related molecules. In the present study, we show that
the intracellular domain of the Notch1 receptor (Notch1 IC), which is
constitutively active, up-regulates HRT2 expression in
10T½ fibroblasts. Luciferase reporter assays using the
regulatory regions of the mouse HRT genes revealed that
transcription of all three genes is stimulated by Notch1 IC. The
promoters of the HRT genes share homology in a binding
site for Suppressor of Hairless [Su(H)], a transcriptional mediator
of Notch signaling. A dominant-negative Su(H) mutant abolished
Notch-activated HRT2 expression, and mutation of the
conserved Su(H) consensus site in the HRT2 promoter
attenuated transcriptional activation by Notch. Ectopic expression of
HRT proteins also blocked activation of HRT2 expression
by Notch1 IC through a mechanism requiring the basic region...
Although an excitotoxic mechanism of neuronal injury has been proposed to play a role in chronic neurodegenerative disorders such as Alzheimer’s disease, and neurotrophic factors have been put forward as potential therapeutic agents, direct evidence is lacking. Taking advantage of the fact that mutations in the presenilin-1 (PS1) gene are causally linked to many cases of early-onset inherited Alzheimer’s disease, we generated PS1 mutant knock-in mice and directly tested the excitotoxic and neurotrophic hypotheses of Alzheimer’s disease. Primary hippocampal neurons from PS1 mutant knock-in mice exhibited increased production of amyloid β-peptide 42/43 and increased vulnerability to excitotoxicity, which occurred in a gene dosage-dependent manner. Neurons expressing mutant PS1 exhibited enhanced calcium responses to glutamate and increased oxyradical production and mitochondrial dysfunction. Pretreatment with either basic fibroblast growth factor or activity-dependent neurotrophic factor protected neurons expressing mutant PS1 against excitotoxicity. Both basic fibroblast growth factor and activity-dependent neurotrophic factor stabilized intracellular calcium levels and abrogated the increased oxyradical production and mitochondrial dysfunction otherwise caused by the PS1 mutation. Our data indicate that neurotrophic factors can interrupt excitotoxic neurodegenerative cascades promoted by PS1 mutations.
Interactions among transcription factors that bind to separate sequence elements require bending of the intervening DNA and juxtaposition of interacting molecular surfaces in an appropriate orientation. Here, we examine the effects of single amino acid substitutions adjacent to the basic regions of Fos and Jun as well as changes in sequences flanking the AP-1 site on DNA bending. Substitution of charged amino acid residues at positions adjacent to the basic DNA-binding domains of Fos and Jun altered DNA bending. The change in DNA bending was directly proportional to the change in net charge for all heterodimeric combinations between these proteins. Fos and Jun induced distinct DNA bends at different binding sites. Exchange of a single base pair outside of the region contacted in the x-ray crystal structure altered DNA bending. Substitution of base pairs flanking the AP-1 site had converse effects on the opposite directions of DNA bending induced by homodimers and heterodimers. These results suggest that Fos and Jun induce DNA bending in part through electrostatic interactions between amino acid residues adjacent to the basic region and base pairs flanking the AP-1 site. DNA bending by Fos and Jun at inverted binding sites indicated that heterodimers bind to the AP-1 site in a preferred orientation. Mutation of a conserved arginine within the basic regions of Fos and transversion of the central C:G base pair in the AP-1 site to G:C had complementary effects on the orientation of heterodimer binding and DNA bending. The conformational variability of the Fos–Jun–AP-1 complex may contribute to its functional versatility at different promoters.
The Arabidopsis thaliana NPR1 has been shown to be a key regulator of gene expression during the onset of a plant disease-resistance response known as systemic acquired resistance. The npr1 mutant plants fail to respond to systemic acquired resistance-inducing signals such as salicylic acid (SA), or express SA-induced pathogenesis-related (PR) genes. Using NPR1 as bait in a yeast two-hybrid screen, we identified a subclass of transcription factors in the basic leucine zipper protein family (AHBP-1b and TGA6) and showed that they interact specifically in yeast and in vitro with NPR1. Point mutations that abolish the NPR1 function in A. thaliana also impair the interactions between NPR1 and the transcription factors in the yeast two-hybrid assay. Furthermore, a gel mobility shift assay showed that the purified transcription factor protein, AHBP-1b, binds specifically to an SA-responsive promoter element of the A. thaliana PR-1 gene. These data suggest that NPR1 may regulate PR-1 gene expression by interacting with a subclass of basic leucine zipper protein transcription factors.
The extracellularly regulated kinase (ERK), one of the three types of mitogen-activated kinases, was rapidly activated after cutting porcine articular cartilage either when maintained as explants or in situ. Cutting released a soluble ERK-activating factor from the cartilage, which was purified and identified by MS as basic fibroblast growth factor (bFGF). Experiments with neutralizing Abs to bFGF and an FGFR1 tyrosine kinase inhibitor showed that this growth factor was the major ERK-activating factor released after injury. Treating cartilage with the heparin-degrading enzyme heparitinase also caused release of bFGF, suggesting the presence of an extracellular store that is sequestered in the matrix and released upon damage. Basic FGF induced the synthesis of a number of chondrocyte proteins including matrix metalloproteinases 1 and 3, tissue inhibitor of metalloproteinases-1, and glycoprotein 38, which were identified by MS. The strong induction of matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 suggests that bFGF could have a role in remodeling damaged tissue.
The biological effects of estrogens are mediated by the estrogen receptors ERα and ERβ. These receptors regulate gene expression through binding to DNA enhancer elements and subsequently recruiting factors such as coactivators that modulate their transcriptional activity. Here we show that ARNT (aryl hydrocarbon receptor nuclear translocator), the obligatory heterodimerization partner for the aryl hydrocarbon receptor and hypoxia inducible factor 1α, functions as a potent coactivator of ERα- and ERβ- dependent transcription. The coactivating effect of ARNT depends on physical interaction with the ERs and involves the C-terminal domain of ARNT and not the structurally conserved basic helix–loop–helix and PAS (Per-ARNT-Sim) motifs. Moreover, we show that ARNT/ER interaction requires the E2-activated ligand binding domain of ERα or ERβ. These observations, together with the previous role of ARNT as an obligatory partner protein for conditionally regulated basic helix–loop–helix–PAS proteins like the aryl hydrocarbon receptor or hypoxia inducible factor 1α, expand the cellular functions of ARNT to include regulation of ERα and ERβ transcriptional activity. ARNT was furthermore recruited to a natural ER target gene promoter in a estrogen-dependent manner...
A significant number of self-reactive T cell clones escape thymic negative
selection and are released into the periphery, where some are potentially
pathogenic. The clonal expansion of self-reactive T cells is known to be
limited during initial antigen encounter by apoptotic or anergic mechanisms,
regulatory CD4+ T cells, and cytokines. Here we report that
superimposed on these mechanisms, during the evolution of autoimmunity in
experimental autoimmune encephalomyelitis (EAE), CD8+ T cells are
induced, which fine-tune the peripheral self-reactive T cell receptor (TCR)
repertoire. We assayed the myelin basic protein-reactive TCR repertoire in
naive, EAE-recovered mice as well as EAE-recovered mice depleted of
CD8+ T cells by TCRVβ surface expression,
complementarity-determining region 3 length distribution, and
complementarity-determining region 3 sequencing analysis. In EAE-recovered
mice, certain myelin basic protein-reactive
CD4+Vβ8.2+ clones are significantly decreased and
this decrease is not observed if CD8+ T cells were depleted from
these mice. The clones that persist in CD8+ T cell-intact mice are
highly diverse in contrast to the clones expanded in CD8+ T
cell-depleted mice, which are dominated by the significant outgrowth of a few
Biological responses to oxygen availability play important roles in development, physiological homeostasis, and many disease processes. In mammalian cells, this adaptation is mediated in part by a conserved pathway centered on the hypoxia-inducible factor (HIF). HIF is a heterodimeric protein complex composed of two members of the basic helix–loop–helix Per-ARNT-Sim (PAS) (ARNT, aryl hydrocarbon receptor nuclear translocator) domain family of transcriptional activators, HIFα and ARNT. Although this complex involves protein–protein interactions mediated by basic helix–loop–helix and PAS domains in both proteins, the role played by the PAS domains is poorly understood. To address this issue, we have studied the structure and interactions of the C-terminal PAS domain of human HIF-2α by NMR spectroscopy. We demonstrate that HIF-2α PAS-B binds the analogous ARNT domain in vitro, showing that residues involved in this interaction are located on the solvent-exposed side of the HIF-2α central β-sheet. Mutating residues at this surface not only disrupts the interaction between isolated PAS domains in vitro but also interferes with the ability of full-length HIF to respond to hypoxia in living cells. Extending our findings to other PAS domains...
Membrane binding of Gag, a crucial step in HIV-1 assembly, is facilitated by bipartite signals within the matrix (MA) domain: N-terminal myristoyl moiety and the highly basic region (HBR). We and others have shown that Gag interacts with a plasma-membrane-specific acidic phospholipid, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], via the HBR, and that this interaction is important for efficient membrane binding and plasma membrane targeting of Gag. Generally, in protein–PI(4,5)P2 interactions, basic residues promote the interaction as docking sites for the acidic headgroup of the lipid. In this study, toward better understanding of the Gag–PI(4,5)P2 interaction, we sought to determine the roles played by all of the basic residues in the HBR. We identified three basic residues promoting PI(4,5)P2-dependent Gag-membrane binding. Unexpectedly, two other HBR residues, Lys25 and Lys26, suppress membrane binding in the absence of PI(4,5)P2 and prevent promiscuous intracellular localization of Gag. This inhibition of nonspecific membrane binding is likely through suppression of myristate-dependent hydrophobic interaction because mutating Lys25 and Lys26 enhances binding of Gag with neutral-charged liposomes. These residues were reported to bind RNA. Importantly...
Nesse, Randolph M.; Bergstrom, Carl T.; Ellison, Peter T.; Flier, Jeffrey S.; Gluckman, Peter; Govindaraju, Diddahally R.; Niethammer, Dietrich; Omenn, Gilbert S.; Perlman, Robert L.; Schwartz, Mark D.; Thomas, Mark G.; Stearns, Stephen C.; Valle, David
Fonte: National Academy of SciencesPublicador: National Academy of Sciences
New applications of evolutionary biology in medicine are being discovered at an accelerating rate, but few physicians have sufficient educational background to use them fully. This article summarizes suggestions from several groups that have considered how evolutionary biology can be useful in medicine, what physicians should learn about it, and when and how they should learn it. Our general conclusion is that evolutionary biology is a crucial basic science for medicine. In addition to looking at established evolutionary methods and topics, such as population genetics and pathogen evolution, we highlight questions about why natural selection leaves bodies vulnerable to disease. Knowledge about evolution provides physicians with an integrative framework that links otherwise disparate bits of knowledge. It replaces the prevalent view of bodies as machines with a biological view of bodies shaped by evolutionary processes. Like other basic sciences, evolutionary biology needs to be taught both before and during medical school. Most introductory biology courses are insufficient to establish competency in evolutionary biology. Premedical students need evolution courses, possibly ones that emphasize medically relevant aspects. In medical school...
The mitochondrial DEAD-box proteins Mss116p of Saccharomyces cerevisiae and CYT-19 of Neurospora crassa are ATP-dependent helicases that function as general RNA chaperones. The helicase core of each protein precedes a C-terminal extension and a basic tail, whose structural role is unclear. Here we used small-angle X-ray scattering to obtain solution structures of the full-length proteins and a series of deletion mutants. We find that the two core domains have a preferred relative orientation in the open state without substrates, and we visualize the transition to a compact closed state upon binding RNA and adenosine nucleotide. An analysis of complexes with large chimeric oligonucleotides shows that the basic tails of both proteins are attached flexibly, enabling them to bind rigid duplex DNA segments extending from the core in different directions. Our results indicate that the basic tails of DEAD-box proteins contribute to RNA-chaperone activity by binding nonspecifically to large RNA substrates and flexibly tethering the core for the unwinding of neighboring duplexes.
Translational research generally refers to the application of knowledge generated by advances in basic sciences research translated into new approaches for diagnosis, prevention, and treatment of disease. This direction is called bench-to-bedside. Psychiatry has similarly emphasized the basic sciences as the starting point of translational research. This article introduces the term translational epidemiology for psychiatry research as a bidirectional concept in which the knowledge generated from the bedside or the population can also be translated to the benches of laboratory science. Epidemiologic studies are primarily observational but can generate representative samples, novel designs, and hypotheses that can be translated into more tractable experimental approaches in the clinical and basic sciences. This bedside-to-bench concept has not been explicated in psychiatry, although there are an increasing number of examples in the research literature. This article describes selected epidemiologic designs, providing examples and opportunities for translational research from community surveys and prospective, birth cohort, and family-based designs. Rapid developments in informatics, emphases on large sample collection for genetic and biomarker studies...
KIST Medical College follows the curriculum of the Institute of Medicine, Tribhuvan University. The programme aims to produce socially responsible and competent physicians who are willing and able to meet the existing and emerging challenges of the national and international healthcare system. The first cohort of undergraduate medical students (MBBS) students was admitted in November 2008 and three cohorts including the one admitted in 2008 have been admitted at the time of writing. The basic science subjects are taught in an integrated, organ-system-based manner with community medicine during the first two years. I was appointed as the MBBS Phase I programme coordinator in September 2008 and in this article I share my experiences of running the basic sciences programme and also offer suggestions for running an efficient academic programme. The manuscript will be of special interest to readers running undergraduate medical programmes. The reader can understand our experiences in running the programme in adverse circumstances, learning to achieve greater integration among basic science, community medicine and clinical departments, obtain information about a community diagnosis programme and know about running special modules on the medical humanities and pharmaceutical promotion.
Well known difficulties of teaching and research lived by basic sciences of the medical school curriculum in Portugal are becoming desperate. Two main problems deserve major concern and urgent solutions: firstly, the recruitment and continuing formation of motivated candidates for teaching and/or research in those areas; secondly, the establishment of adequate economic support that makes those careers in basic teaching and research attractive, along with the provision or creation of the necessary support for both activities. Specific administrative and financial solutions are required from the government, in order to foster a realistic future in the preclinical areas of the medical curricula.; Well known difficulties of teaching and research lived by basic sciences of the medical school curriculum in Portugal are becoming desperate. Two main problems deserve major concern and urgent solutions: firstly, the recruitment and continuing formation of motivated candidates for teaching and/or research in those areas; secondly, the establishment of adequate economic support that makes those careers in basic teaching and research attractive, along with the provision or creation of the necessary support for both activities. Specific administrative and financial solutions are required from the government...
The author analyses the problems involved in the transfer of concepts and techniques from the basic sciences to the medical practice. The number of skills to be acquired in the course of medical training restricts the inclusion of the teaching in depth of basic sciences in the Curricula of medical schools. Post-graduate teaching cannot be used for that purpose unless it is geared to MD's fully dedicated to research. New discoveries in the basic sciences which may be relevant to medicine can only be made available to doctors through the development of basic research in medical institutions, playing the role of centers of excellence. Since basic research in the medical field is, by its nature, interdisciplinary, such policy entails the recruitment of scientists with non-medical background to whom career opportunities comparable to those of the doctors should be made available. In countries with small scientific communities the most important step in the stimulation of basic research is the identification of young talents and the support of existing productive groups. In the mid term, molecular biology, neurobiology and signal processing (specially image processing) seem to be promising areas from a medical point of view. Science should be considered a very important component of the cultural activity of a country.; The author analyses the problems involved in the transfer of concepts and techniques from the basic sciences to the medical practice. The number of skills to be acquired in the course of medical training restricts the inclusion of the teaching in depth of basic sciences in the Curricula of medical schools. Post-graduate teaching cannot be used for that purpose unless it is geared to MD's fully dedicated to research. New discoveries in the basic sciences which may be relevant to medicine can only be made available to doctors through the development of basic research in medical institutions...
The medial olivocochlear reflex (MOCR) is a brainstem-based neural feedback circuit by which mammals adaptively adjust the gain of their ears in response to changing environmental conditions. Activating the reflex with sound reduces cochlear gain, but the mechanisms by which the reflex produces its cochlear effects, the role(s) the reflex plays in hearing and many basic reflex properties are not well-understood. This thesis quantifies four basic properties of the reflex in humans using stimulus-frequency-otoacoustic-emissions (SFOAEs) that address the following issues: (1) The relative strengths of ipsilateral and contralateral reflex pathways (2) The reflex time-course (3) The response of the reflex to amplitude modulated (AM) noise (4;) The distribution of reflex strengths across a normal-hearing population Activating the reflex with ipsilateral or contralateral noise produced, on average, the same effect in cochlea at the 1 kHz place, contrary to expectations based upon animal studies. Simultaneous bilateral activation produced an effect that was equivalent to the sum of ipsilateral and contralateral activations, on average. Thus, no prevailing binaural interaction took place for our stimulus. Activating the reflex caused detectable changes in the cochlea within 25 ms; the changes continued to develop for 100's of milliseconds.; (cont.) The decay rate upon reflex deactivation was generally faster than the onset rate ([tau]decay= 159 ± 54 ms...
The history of cardiology encompasses some of the most revered names in medical history, many belonging to physicians who have advanced knowledge beyond their time. However, there have been countless others whose work in the basic sciences has paid large dividends to clinical cardiology. The original example of such an individual is William Harvey, whose reasoned experimentation led to the understanding of the circulation of blood. Another such man, Sir James Black, has contributed to basic scientific and clinical knowledge in cardiology, both as a physician and as a basic scientist. His invention of propranolol, the beta adrenergic receptor antagonist that revolutionized the medical management of angina pectoris, is considered to be one of the most important contributions to clinical medicine and pharmacology of the 20th century. His method of research, his discoveries about adrenergic pharmacology, and his clarification of the mechanisms of cardiac action are all strengths of his work. In 1988, he was awarded the Nobel Prize in Medicine. Sir James's conclusions and method of research have continued to influence work in clinical pharmacology and cardiovascular medicine. Thus, the development of propranolol runs parallel to most other great achievements in medicine: the genius of a few builds on the accomplishments of many...
The quantity and quality of scientific output of the topmost 50 countries in
the four basic sciences (agricultural and biological sciences, chemistry,
mathematics, and physics and astronomy) are studied in the period of the recent
12 years (1996-2007). In order to rank the countries, a novel two-dimensional
method is proposed, which is inspired by the H-index and other methods based on
quality and quantity measures. The countries data are represented in a
"quantity-quality diagram", and partitioned by a conventional statistical
algorithm (k-means), into three clusters, members of which are rather the same
in all of the basic sciences. The results offer a new perspective on the global
positions of countries with regards to their scientific output.; Comment: 6 pages, 5 figures
In recent decades it has been detected in Italy a decrease in enrollment in
basic sciences, i.e. Mathematics, Physics and Chemistry. The increase in
specific orientation is strategically crucial to achieve the goal of
maintaining and increasing the number of motivated and capable students who
enroll in these courses. With the purpose of increasing scientific vocations,
workshops were organized in high schools and teachers involved in planning and
implementation of laboratories, conferences for scientific outreach, thematic
exhibitions, guided tours of research laboratories, summer's schools for
students and courses for teachers were realized for developing a cultural
enhancement in teaching basic sciences. Particularly significant is the case of
activities organized by the Department of Physics of the University of Siena
for students and teachers in Southern Tuscany. The methods used in cultural
enhancement of teachers and activities designed to support schools with limited
laboratory facilities, together with stimulating activities for motivated
students are allowed to take root for some good practices in physics teaching
and orientation to scientific degrees. Beyond describing the main activities
for orientation to Physics, activities done in partnership with chemists...