The temporal impulse response function in infantile nystagmus.

Despite rapid to-and-fro motion of the retinal image that results from their incessant involuntary eye movements, persons with infantile nystagmus (IN) rarely report the perception of motion smear. We performed two experiments to determine if the reduction of perceived motion smear in persons with IN is associated with an increase in the speed of the temporal impulse response. In Experiment 1, increment thresholds were determined for pairs of successively presented flashes of a long horizontal line, presented on a 65-cd/m2 background field. The stimulus-onset asynchrony (SOA) between the first and second flash varied from 5.9 to 234 ms. In experiment 2, temporal contrast sensitivity functions were determined for a 3-cpd horizontal square-wave grating that underwent counterphase flicker at temporal frequencies between 1 and 40 Hz. Data were obtained for 2 subjects with predominantly pendular IN and 8 normal observers in Experiment 1 and for 3 subjects with IN and 4 normal observers in Experiment 2. Temporal impulse response functions (TIRFs) were estimated as the impulse response of a linear second-order system that provided the best fit to the increment threshold data in Experiment 1 and to the temporal contrast sensitivity functions in Experiment 2. Estimated TIRFs of the subjects with pendular IN have natural temporal frequencies that are significantly faster than those of normal observers (ca. 13 vs. 9 Hz), indicating an accelerated temporal response to visual stimuli. This increase in response speed is too small to account by itself for the virtual absence of perceived motion smear in subjects with IN, and additional neural mechanisms are considered.

Proteomic identification of in vivo substrates for matrix metalloproteinases 2 and 9 reveals a mechanism for resolution of inflammation.

Clearance of allergic inflammatory cells from the lung through matrix metalloproteinases (MMPs) is necessary to prevent lethal asphyxiation, but mechanistic insight into this essential homeostatic process is lacking. In this study, we have used a proteomics approach to determine how MMPs promote egression of lung inflammatory cells through the airway. MMP2- and MMP9-dependent cleavage of individual Th2 chemokines modulated their chemotactic activity; however, the net effect of complementing bronchoalveolar lavage fluid of allergen-challenged MMP2(-/-)/MMP9(-/-) mice with active MMP2 and MMP9 was to markedly enhance its overall chemotactic activity. In the bronchoalveolar fluid of MMP2(-/-)/MMP9(-/-) allergic mice, we identified several chemotactic molecules that possessed putative MMP2 and MMP9 cleavage sites and were present as higher molecular mass species. In vitro cleavage assays and mass spectroscopy confirmed that three of the identified proteins, Ym1, S100A8, and S100A9, were substrates of MMP2, MMP9, or both. Function-blocking Abs to S100 proteins significantly altered allergic inflammatory cell migration into the alveolar space. Thus, an important effect of MMPs is to differentially modify chemotactic bioactivity through proteolytic processing of proteins present in the airway. These findings provide a molecular mechanism to explain the enhanced clearance of lung inflammatory cells through the airway and reveal a novel approach to target new therapies for asthma.

Identification of two vitamin D response elements in the rat osteopontin gene

Studies to elucidate the function of vitamin D have demonstrated an important role in regulating bone-related cells, including osteoblasts and osteoclasts. A seemingly paradoxical observation is that 1,25(OH)$\sb2$D$\sb3$, the active metabolite of vitamin D, stimulates bone resorption, yet regulates transcription of genes expressed by osteoblasts. One mechanism that could explain these actions is the upregulation of transcription of osteoblast-specific genes. These gene products could then act as effectors to influence osteoclastic activity. We hypothesized that molecular signals could be deposited directly into the mineralized matrix in the form of noncollagenous proteins, such as osteopontin (OPN). The structure, biosynthesis and localization of OPN suggest that it could function to mediate the molecular "cross talk" between osteoblasts and osteoclasts in response to 1,25(OH)$\sb2$D$\sb3$. To begin to address this hypothesis, elucidation of the molecular mechanisms of action involved in the transactivation of OPN by 1,25(OH)$\sb2$D$\sb3$ is essential.^ In the present study, the rat opn gene was isolated and characterized. Functional analysis by transient transfection of the 5$\sp\prime$ flanking sequences of the rat opn gene fused to the luciferase gene demonstrated that OPN is transcriptionally upregulated by 1,25(OH)$\sb2$D$\sb3$, mediated through two vitamin D response elements (VDRE). Both proximal and distal VDREs are structurally similar (two imperfect direct repeats separated by a 3 nucleotide spacer) and bind protein complexes that include the VDR and retinoid-X receptor (RXR). Isolated VDRE expression constructs produce functional activity of equivalent magnitude of responsiveness to 1,25(OH)$\sb2$D$\sb3$. However, expression constructs containing either VDRE and at least 200 bp of 5$\sp\prime$ and 3$\sp\prime$ flanking sequence demonstrated that the distal VDRE produces an amplitude of response significantly higher than the proximal VDRE. We conclude that the transcriptional upregulation of the opn gene by 1,25(OH)$\sb2$D$\sb3$ involves the transactivation of two VDREs, while maximal responsiveness requires interaction of the VDREs with additional cis-elements contained in the 5$\sp\prime$ sequence. ^

LOCALIZATION OF DNA REPAIR FUNCTIONS TO THE NUCLEAR MATRIX

The nucleus of a eukaryotic cell contains both structural and functional elements that contribute to the controlled operation of the cell. In this context, functional components refers to those nuclear constituents that perform metabolic activities such as DNA replication and RNA transcription. Structural nuclear components, designated nuclear matrix, organize the DNA into loops or domains and appear to provide a framework for nuclear DNA organization. However, the boundary between structural and functional components is not clear cut as evinced by reports of associations between metabolic functions and the nuclear matrix. The studies reported here attempt to determine the relationship of another nuclear function, DNA repair, to the nuclear matrix.^ One objective of these studies was to study the initiation of DNA repair by directly measuring the UV-incision activities in human cells and determine the influence of various extractable nuclear components on these activities. The assay for incision activities required the development of a nuclear isolation protocol that produced nuclei with intact DNA; the conformation of the nuclear DNA and its physical characteristics in response to denaturing conditions were determined.^ The nuclei produced with this protocol were then used as substrates for endogenous UV-specific nuclease activities. The isolated nuclei were shown to contain activities that cause breaks in nuclear DNA in response to UV-irradiation. These UV-responsive activities were tightly associated with nuclear components, being unextractable with salt concentration of up to 0.6 M.^ The tight association of the incision activities with salt-extracted nuclei suggested that other repair function might also be associated with salt-stable components of the nucleus. The site of unscheduled DNA synthesis (UDS) was determined in salt-extracted nuclei (nucleoids) using autoradiography and fluorescent microscopy. UDS was found to occur in association with the nuclear matrix following low-doses (2.55 J/M('2)) of ultraviolet light, but the association became looser after higher doses of ultraviolet light (10-30 J/m('2)). ^

Retinoid -induced regression of human head and neck squamous cell carcinomas is associated with the induction of a pro -inflammatory response

Retinoids are Vitamin A derivatives that are effective chemopreventative and chemotherapeutic agents for head and neck squamous cell carcinomas (HNSCC). Despite the wide application of retinoids in cancer treatment, the mechanism by which retinoids inhibit head and neck squamous cell carcinomas is not completely understood. While in vitro models show that drugs affect cell proliferation and differentiation, in vivo models, such as tumor xenografts in nude mice drugs affect more complex parameters such as extracellular matrix formation, angiogenesis and inflammation. Therefore, we studied the effects of retinoids on the growth of the 22B HNSCC tumors using a xenograft model. In this system, retinoids had no effect on tumor cell differentiation but caused invasion of the tumor by inflammatory cells. Retinoid induced inflammation lead to tumor cell death and tumor regression. Therefore, we hypothesized that retinoids stimulated the 22B HNSCC xenografts to produce a pro-inflammatory signal such as chemokines that in turn activated host inflammatory responses. ^ We used real time quantitative RT-PCR to measure cytokine and chemokine expression in retinoid treated tumors. Treatment of tumors with an RAR-specific retinoid, LGD1550, had no effect on the expression of TNFα, IL-1α, GROα, IP-10, Rantes, MCP-1 and MIP-1α but induced IL-8 mRNA 5-fold. We further characterized the retinoid effect on IL-8 expression on the 22B HNSCC and 1483 HNSCC cells in vitro. Retinoids increased IL-8 expression and enhanced TNFα-dependent IL-8 induction. In addition, retinoids increased the basal and TNFα-dependent expression of MCP-1 but decreased the basal and TNFα dependent expression of IP-10. The effect of retinoids on IL-8 and MCP-1 expression was very rapid with increased levels of mRNA detected within 1–2 hours. This effect did not require new protein synthesis and did not result from mRNA stabilization. Both RAR and RXR ligands increased IL-8 expression whereas only RAR ligands activated MCP-1 expression. ^ We identified a functional retinoid response element in the IL-8 promoter that was located adjacent to the C/EBP-NFkB response element. TNFα treatment of the 22B cells caused rapid, transient and selective acetylation of regions of the IL-8 promoter associated with the NFkB response element. Co-treatment of the cells with retinoids plus TNF increased the acetylation of chromatin in this region without altering the kinetics of acetylation. These results demonstrate that ligand activated retinoid receptors can cooperate with NFkB in histone acetylation and chromatin remodeling. We believe that in certain HNSCC tumors this cooperation and the resulting enhancement of IL-8 expression can induce an inflammatory response that leads to tumor regression. We believe that the induction of inflammation in susceptible tumors, possibly coupled with cytotoxic interventions may be an important component in the use of retinoids to treat human squamous cancers. ^

Activation of matrix metalloproteinase -9 (MMP-9) by heregulin in human breast cancer cells involves multiple signaling pathways

Matrix metalloproteinase-9 (MMP-9) plays an important role in tumor invasion and angiogenesis. Secretion of MMP-9 has been reported in various cancer types including lung cancer, brain cancer, colon cancer, and breast cancer. Heregulin is a growth factor that regulates growth and differentiation of normal breast cells as well as mammary tumor cells. To study the role of heregulin in breast cancer metastasis, we tested whether heregulin may regulate MMP-9 secretion. By screening a panel of breast cancer cell line for their ability to respond to heregulin and produce MMP-9, we have found that MMP-9 secretion can be induced by heregulin-β1 in two breast cancer cell lines, SKBr3 and MCF-7. In both cell lines, increase of MMP-9 activity as shown by zymography was accompanied by increased protein level as well as mRNA level of MMP-9. Using a reporter luciferase assay, we have identified that proximal −670bp promoter of MMP-9 had similar activity to a 2.2kb MMP-9 promoter in response to heregulin stimulation. Heregulin treatment of SKBr3 and MCF-7 activated multiple signaling pathways inside cells. These include the Erk pathway, the p38 kinase pathway, PKC pathway, and PI-3K pathway. To examine which pathways are involved in MMP-9 activation by heregulin, we have used a panel of chemical inhibitors to specifically inhibit each one of these pathways. Ro-31-8220 (PKC inhibitor) and SB203580 (p38 kinase inhibitor) completely blocked heregulin activation of MMP-9. On the other hand, PD098059 (MEK-1 inhibitor) partially blocked MMP-9 activation, whereas PI-3K inhibitor, wortmannin, had no effect. Therefore, at least three signaling pathways are involved in activation of MMP-9 by heregulin. Since MMP-9 is tightly associated with metastatic potential, our study also suggests that heregulin may enhance breast tumor metastasis through induction of MMP-9 expression. ^