Abstract. Sphingosine, a metabolite of membrane sphingolipids, regulates proliferation of quiescent Swiss 3T3 fibroblasts (Zhang, H ., N. E. Buckley, K. Gibson, and S. Spiegel . 1990. J. Biol. Chem. 265:76-81) . The present study provides new insights into the formation and function of a unique phospholipid, a metabolite of sphingosine, which was unequivocally identified as sphingosine-l-phosphate . The rapid increase in 32P-labeled sphingosine-l-phosphate levels induced by sphingosine was concentration dependent and correlated with its effect on DNA synthesis . Similar to the mitogenic effects of sphingosine, low concentrations ofsphingosine-1-phosphate stimulated DNA synthesis and induced pronounced morphological alterations . Both sphingosine and sphingosine-l-phosphate stimulated DNA synthesis in cells made protein kinase C deficient by prolonged treatment with phorbol ester and sphingosine still T HE biochemical mechanisms whereby eukaryotic cells regulate their proliferation are not well understood . One approach to this problem is to identify the second messengers responsible for the initiation of the progression of Góarrested cells into S phase. In Swiss 3T3 cells, which are sensitive to a wide range of mitogenic agents, some growth factors appear to function through conventional second messengers such as cAMP, whereas others use the signal pathways associated with increased degradation ofpolyphosphoinositides leading to the generation of lipid second messengers. Diacylglycerol (DAG)' is an endogenous activator of protein kinase C (Nishizuka, 1986) and inositol trisphosphate (IP3) causes a release ofCal+ from intracellular stores (Berridge, 1984;Berridge et al., 1984) . Although the roles of these intracellular second messengers which appear to be important for the mitogenic response have been well characterized, it is evident that not all of the second messenger sys-

We present a new method for recording digital holograms under incoherent illumination. Light is reflected from a 3D object, propagates through a diffractive optical element (DOE), and is recorded by a digital camera. Three holograms are recorded sequentially, each for a different phase factor of the DOE. The three holograms are superposed in the computer, such that the result is a complex-valued Fresnel hologram. When this hologram is reconstructed in the computer, the 3D properties of the object are revealed.

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Fresnel Incoherent Correlation Holography (FINCH) enables holograms to be recorded from incoherent light with just a digital camera and spatial light modulator. We previously described its application to general three dimensional incoherent imaging and specifically to fluorescence microscopy, wherein one complex hologram contains the three dimensional information in the field of view, obviating the need for scanning or serial sectioning. We have now further analyzed FINCH in view of linear system theory and in comparison to conventional coherent and incoherent two dimensional imaging systems. We demonstrate, theoretically and experimentally, improved resolution by FINCH, when compared to conventional imaging.

Interleukin-10 (IL-10) has been shown to reduce neuronal degeneration after CNS injury. However, the molecular mechanisms underlying the neuroprotective properties of this cytokine are still under investigation. Glutamate exacerbates secondary injury caused by trauma. Thus, we examined whether IL-10 prevents glutamate-mediated cell death. We used rat cerebellar granule cells in culture because these neurons undergo apoptosis upon exposure to toxic concentrations of glutamate (100-500 microm) or NMDA (300 microm). Pretreatment of cerebellar granule cells with IL-10 (1-50 ng/ml) elicited a dose- and time-dependent reduction of glutamate-induced excitotoxicity. Most importantly, IL-10 reduced the number of apoptotic cells when added to the cultures together or 1 hr after glutamate. Using patch-clamping and fluorescence Ca(2+) imaging techniques, we examined whether IL-10 prevents glutamate toxicity by blocking the function of NMDA channel. IL-10 failed to affect NMDA channel properties and to reduce NMDA-mediated rise in intracellular Ca(2+). Thus, this cytokine appears to prevent glutamate toxicity by a mechanism unrelated to a blockade of NMDA receptor function. Various proteases, such as caspase-3, and transcription factors, such as nuclear factor kappaB (NF-kappaB), have been proposed to participate in glutamate-mediated apoptosis. Thus, we examined whether IL-10 modulates the activity of these apoptotic markers. IL-10 blocked both the glutamate-mediated induction of caspase-3 as well as NF-kappaB DNA binding activity, suggesting that the neuroprotective properties of IL-10 may rely on its ability to block the activity of proapoptotic proteins.

Fresnel Incoherent Correlation Holography (FINCH) enables holograms and 3D images to be created from incoherent light with just a camera and spatial light modulator (SLM). We previously described its application to microscopic incoherent fluorescence wherein one complex hologram contains all the 3D information in the microscope field, obviating the need for scanning or serial sectioning. We now report experiments which have led to the optimal optical, electro-optic, and computational conditions necessary to produce holograms which yield high quality 3D images from fluorescent microscopic specimens. An important improvement from our previous FINCH configurations capitalizes on the polarization sensitivity of the SLM so that the same SLM pixels which create the spherical wave simulating the microscope tube lens, also pass the plane waves from the infinity corrected microscope objective, so that interference between the two wave types at the camera creates a hologram. This advance dramatically improves the resolution of the FINCH system. Results from imaging a fluorescent USAF pattern and a pollen grain slide reveal resolution which approaches the Rayleigh limit by this simple method for 3D fluorescent microscopic imaging.

Fresnel incoherent correlation holography (FINCH) records holograms under incoherent illumination. FINCH was implemented with two focal length diffractive lenses on a spatial light modulator (SLM). Improved image resolution over previous single lens systems and at wider bandwidths was observed. For a given image magnification and light source bandwidth, FINCH with two lenses of close focal lengths yields a better hologram in comparison to a single diffractive lens FINCH. Three techniques of lens multiplexing on the SLM were tested and the best method was randomly and uniformly distributing the two lenses. The improved quality of the hologram results from a reduced optical path difference of the interfering beams and increased efficiency.

We present a new imaging method to record multicolor digital holograms from objects emitting fluorescent light. The fluorescent light specific to the emission wavelength of various fluorescent dyes after excitation of three dimensional (3D) objects is recorded on a digital monochrome camera after reflection from a diffractive optical element (DOE). For each wavelength of fluorescent emission, the camera sequentially records three holograms reflected from the DOE, each with a different phase factor of the DOE's function. The three holograms are superposed in a computer to create a complex valued Fresnel hologram of each fluorescent emission. The holograms for each fluorescent color are further combined in a computer to produce a multicolored fluorescence hologram and 3D color image.