The synapsins are a family of proteins associated with small synaptic vesicles that are implicated in synaptic maintenance and in the supply of vesicles for exocytosis. They are well characterized as substrates for protein kinases, and one class of synapsin, synapsin I, has been shown to bind, and be regulated by, calmodulin. A representative of the synapsin II class is now shown to bind calmodulin. Optical biosensor assays of Ca2+-dependent calmodulin binding to recombinant rat synapsin IIb indicated an apparent KD for calmodulin of 31 +/- 5 nM. Phosphorylation at Ser 10 increased the rates of calmodulin association (by a factor of 10) and dissociation (by a factor of 20). Fragment analysis and predictions from the sequence indicated two potential calmodulin binding sequences in the conserved central (C) domain. Peptides representing these sequences (residues 122-143 and 313-334 in synapsin IIb) were synthesized. Peptide 122-143 was found to bind calmodulin (KD 32 +/- 10 nM) and inhibit interaction of synapsin IIb with calmodulin. The interaction of peptide 313-334 was much weaker. Sequences similar to residues 122-143 are present in all published synapsin sequences. Calmodulin binding by synapsins seems not to be confined to mammals: a recombinant Drosophila synapsin 1 fragment containing part of the C-domain showed Ca2+-dependent binding to mammalian calmodulin. We conclude that calmodulin binding to synapsins is likely to be a general aspect of regulation of synaptic function.

Today, over consumption, pollution and resource depletion threaten sustainability. Waste management policies frequently fail to reduce consumption, prevent pollution, conserve resources and foster sustainable products -as seen in the context of managing end-of-life refrigerators and appliances containing ozone-depleting substances (ODSs). However, waste policies are changing to focus on lifecycle impacts of products from the cradle to the grave by extending responsibilities of stakeholders to post-consumer management. Product stewardship and extended producer responsibility are two policies in use, with radically different results when compared for one consumer product, refrigerators. North America has enacted product stewardship policies that fail to require producers to take physical or financial responsibility for recycling or for environmentally sound disposal, so that releases of ozone depleting substances routinely occur, which contribute to the expanding the ozone hole. Conversely, Europe's Waste Electrical and Electronic Equipment (WEEE) Directive requires extended producer responsibility, whereby producers collect and manage their own post-consumer waste products. WEEE has resulted in high recycling rates of greater than 85%, reduced emissions of ODSs and other toxins, greener production methods, such as replacing greenhouse gas refrigerants with environmentally friendly hydrocarbons and more reuse of refrigerators in EU compared to North America.

We previously reported that mesenchymal cells (dermal fibroblasts and dermal papilla cells) can stimulate dopa oxidase activity in the skin melanocytes. This study extends the investigation of the influence of the fibroblast in a comparative study of melanogenesis in melanocytes from the hair, the skin and the eye. Culture of melanocytes with normal proliferative dermal fibroblasts slightly increased dopa oxidase activity of the hair, skin and ocular melanocytes (by 17, 11 and 28%, respectively), but co-culture with fibroblasts recovering from storage in liquid nitrogen or growth-arrested by means of gamma radiation showed much greater effects. Most dramatic results were obtained with fibroblasts, which had been both gamma-irradiated and then frozen in liquid nitrogen, where increases in dopa oxidase activity of 125, 227 and 185% for melanocytes of the hair, the skin and the eye, respectively, were seen. Experiments by using transwell cultures of melanocytes and fibroblasts and by using fibroblast-conditioned medium showed that a large proportion of this fibroblast influence could be mediated by diffusible factors, of which a good proportion was attributable to basic Fibroblast Growth Factor (bFGF). The addition of bFGF significantly increased dopa oxidase activity of the skin melanocytes, when fibroblasts were present, but not in their absence. These data show that fibroblasts in vitro, particularly when deliberately stressed, have the ability to increase dopa oxidase activity in melanocytes of the hair, the skin and the eye and further suggest that this effect is mediated by bFGF acting in combination with some other fibroblast-derived factors.

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The synapsins are a family of four closely related neuronal phosphoproteins localised at the presynaptic nerve terminal [for review see I]. They link small synaptic vesicles to a number of cytoskeletal proteins including actin, tubulin, f o h n and calmodulin. Functions include the regulation of efficiency of neurotransmitter release and they are also implicated in synapse formation. Thus the synapsins are involved in both neurotransmitter exocytosis and neuronal development. In this study, we have prepared a panel of monoclonal antibodies to purified ovine brain synapsin I with a view to using them as probes of synapsin I bindmg functions. Balb/c mice were immunised sub-cutaneously with 10-60 pg at monthly intervals. The protein was emulsified in Freund's complete adjuvant for the first injection and with incomplete adjuvant thereafter. Three days before fusion, the mice were injected intraperitonealy with 60 pg of synapsin I in phosphate buffered saline.Splenocytes were fused with Sp 210 -Ag14 essentially as described previously [2]. The resultant heterokaryotic cells were initially cultured in a HAT medium (RPMI 1640 based medium containing hypoxanthine / aminopterin / thymidme (HAT) and 10 ' YO foetal bovine serum (Gibco)). Initial screening was by enzyme linked immunosorbent assays (ELISAs) on whole synapsin I or the N-terminal head region of the protein produced by collagenase digestion [3]. A large number of positive hybridomas were cloned three times by limited dilution and screened via ELISA. Four highly active antibodies were further screened by Western blotting on whole synapsin I, collagenase-derived head region, or fragments generated by 2-nitro-5-thobenzoic acid (NTCB) cleavage at cysteine residues [4] as shown in figure 1 . Antibodes SNHI and D7EH recognised epitopes contained in the N-terminal portion of the head. SNTl and ClBT were reactive to the C-terminal 'tail' fragments of synapsin I prepared as above (see table 1). No antibodies specific to the 15 kDa mid-synapsin fragment were detected. SNHI, SNTl and D7EH were monospecific for synapsin I and C l B T cross-reacted with five other proteins in an ovine brain homogenate. Immunohistochemistry using SNTl on rat brain sections demonstrated a distinctive pattern of staining predicted for a protein localised at presynaptic nerve terminals. The antibodies also specifically recognised synapsin I in immunoblots of homogenates of rat, cow, rabbit, guinea pig, mouse and human brain. Table 1. Monoclonal antibodies against synapsin 1 Epitope Antibody Ig Class Collagenase fragment NTCB fragments (48 kDa, N-terminal) (31/35 m a ) SNTI IgG I + ClBT IgM + SNHl IgG I + D7EH IeG 1 -L