Cell-specific expression of the insulin gene is controlled by cis-acting DNA sequences located within "-350 base pairs of the 5' flanking DNA immediately upstrVam from the transcription start site. Using synthetic oligonucleotides, we have constructed a systematic series of block replacement mutants spanning this region. No single sequence appears to be absolutely required for expression. However, three of the mutants exhibit 5-10 times less activity and several others show 2-3 times less. Simultaneous mutation of twQ of the most mutationally sensitive regions leads to virtual abolition of activity. These two elements are structurally related and presumably represent key components of the machinery determining the cell-specific expression of the insulin gene.The development of complex multicellular organisms requires selective spatial and temporal regulation of the genetic repertoire. Although cell-specific gene expression in higher eukaryotes can be controlled at multiple levels (1), a primary determinant is the efficiency of initiation of new transcripts (2). Specific transcription is apparently controlled by the interaction of cellular factors with certain cis-acting DNA sequences in the vicinity of the gene.Cis-acting elements involved in controlling cell-specific expression have been identified in several cellular genes. In the insulin gene, two distinct positive control elements have been identified in the 5' flanking DNA (3): a cell-specific enhancer and a cell-specific promoter (4). Additional negatively acting cis elements have also been detected in this region (5). In immunoglobulin genes, cell specificity is also attributable to cell-specific enhancers (6-9) and cell-specific promoters (10-12). A further measure of control may be exerted in this system at the post-transcriptional level (1,12).In vitro binding studies of transcriptional control regions reveal multiple sites for binding of sequence-specific protein factors (13). Cell-specific transcription may be the consequence of the interaction of specific DNA-binding protein factors, which are present at elevated levels in cells actively expressing the specific gene (14-16).To more completely define the elements of the insulin gene that control transcription in vivo and to correlate in vitro protein binding to transcriptional activity, we have constructed a systematic series of block replacement mutants spanning the proximal 345 base pairs (bp) of flanking DNA by using an oligonucleotide-directed approach that permits mutation of a region without changing distances or the stereospecific alignment between transcriptionally important elements. Each mutant contained a block of noncomplementary transversions (i.e., A + C, G ++ T; see ref. 17) spanning -'10 bp. None of these mutations leads to complete loss of activity. However, several produce dramatic reductions (by a factor of 5-10): the "TATA" region of the promoter and two short sequences within the enhancer, which are similar to each other and are also related to the transcriptional control...

Retinoic acid (RA) has been implicated in cardiac morphogenesis by its teratogenic effects on the heart, although its role in normal cardiogenesis remains unknown. To define the parameters of RA action in cardiac morphogenesis, we analyzed the patterns of ligand synthesis, response, and inactivation in the developing mouse heart. Activation of a lacZ transgene controlled by an RA response element (RARE) was compared to the localization of the retinaldehyde-oxidizing dehydrogenase RALDH2, the earliest RA synthetic enzyme in the mouse embryo, and to the expression of a gene encoding an RA-degrading enzyme (P450RA). We observed that RALDH2 localization and RA response were virtually superimposable throughout heart development. Initially, both RALDH2 and RARE-LacZ activity were restricted to the sinus venosa in unlooped hearts, but were high in the dorsal mesocardium, while P450RA expression was restricted to the endocardium. Later stages were characterized by a sequential, noncontiguous progression of RALDH2 accumulation and RA response, from the sinus venosa to atria, dorsal-medial conotruncus, aortic arches, and the epicardium. This dynamic pattern of RA response was a direct result of localized RALDH2, since hearts of cultured embryos were uniformly competent to respond to an exogenous RA challenge. These observations support a model in which the influence of endogenous RA on heart development depends upon localized presentation of the ligand, with only limited diffusion from the source of its synthesis.

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory disease in which adults have significant joint issues leading to poor health. Poor health is compounded by many factors, including exercise avoidance and increased risk of opportunistic infection. Exercise training can improve the health of patients with RA and potentially improve immune function; however, information on the effects of high-intensity interval training (HIIT) in RA is limited. We sought to determine whether 10 weeks of a walking-based HIIT program would be associated with health improvements as measured by disease activity and aerobic fitness. Further, we assessed whether HIIT was associated with improved immune function, specifically antimicrobial/bacterial functions of neutrophils and monocytes.MethodsTwelve physically inactive adults aged 64 ± 7 years with either seropositive or radiographically proven (bone erosions) RA completed 10 weeks of high-intensity interval walking. Training consisted of 3 × 30-minute sessions/week of ten ≥ 60-second intervals of high intensity (80–90% VO2reserve) separated by similar bouts of lower-intensity intervals (50–60% VO2reserve). Pre- and postintervention assessments included aerobic and physical function; disease activity as measured by Disease Activity score in 28 joints (DAS28), self-perceived health, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR); plasma interleukin (IL)-1β, IL-6, chemokine (C-X-C motif) ligand (CXCL)-8, IL-10, and tumor necrosis factor (TNF)-α concentrations; and neutrophil and monocyte phenotypes and functions.ResultsDespite minimal body composition change, cardiorespiratory fitness increased by 9% (change in both relative and absolute aerobic capacity; p < 0.001), and resting blood pressure and heart rate were both reduced (both p < 0.05). Postintervention disease activity was reduced by 38% (DAS28; p = 0.001) with significant reductions in ESR and swollen joints as well as improved self-perceived health. Neutrophil migration toward CXCL-8 (p = 0.003), phagocytosis of Escherichia coli (p = 0.03), and ROS production (p < 0.001) all increased following training. The frequency of cluster of differentiation 14-positive (CD14+)/CD16+ monocytes was reduced (p = 0.002), with both nonclassical (CD14dim/CD16bright) and intermediate (CD14bright/CD16positive) monocytes being reduced (both p < 0.05). Following training, the cell surface expression of intermediate monocyte Toll-like receptor 2 (TLR2), TLR4, and HLA-DR was reduced (all p < 0.05), and monocyte phagocytosis of E. coli increased (p = 0.02). No changes were observed for inflammatory markers IL-1β, IL-6, CXCL-8, IL-10, CRP, or TNF-α.ConclusionsWe report for the first time, to our knowledge, that a high-intensity interval walking protocol in older adults with stable RA is associated with reduced disease activity, improved cardiovascular fitness, and improved innate immune functions, indicative of reduced infection risk and inflammatory potential. Importantly, the exercise program was well tolerated by these ...

OBJECTIVERegenerating organs in diverse biological systems have provided clues to processes that can be harnessed to repair damaged tissue. Adult mammalian β-cells have a limited capacity to regenerate, resulting in diabetes and lifelong reliance on insulin. Zebrafish have been used as a model for the regeneration of many organs. We demonstrate the regeneration of adult zebrafish pancreatic β-cells. This nonmammalian model can be used to define pathways for islet-cell regeneration in humans.RESEARCH DESIGN AND METHODSAdult transgenic zebrafish were injected with a single high dose of streptozotocin or metronidazole and anesthetized at 3, 7, or 14 days or pancreatectomized. Blood glucose measurements were determined and gut sections were analyzed using specific endocrine, exocrine, and duct cell markers as well as markers for dividing cells.RESULTSZebrafish recovered rapidly without the need for insulin injections, and normoglycemia was attained within 2 weeks. Although few proliferating cells were present in vehicles, ablation caused islet destruction and a striking increase of proliferating cells, some of which were Pdx1 positive. Dividing cells were primarily associated with affected islets and ducts but, with the exception of surgical partial pancreatectomy, were not extensively β-cells.CONCLUSIONSThe ability of the zebrafish to regenerate a functional pancreas using chemical, genetic, and surgical approaches enabled us to identify patterns of cell proliferation in islets and ducts. Further study of the origin and contribution of proliferating cells in reestablishing islet function could provide strategies for treating human diseases.