Melanogenesis and melanosome transfer from the melanocytes to the neighboring keratinocytes are induced by ultraviolet radiation and modulated by autocrine and paracrine factors. Keratinocyte growth factor (KGF/fibroblast growth factor (FGF)7) is a paracrine mediator of human keratinocyte growth and differentiation. We evaluated the influence of KGF on melanosome transfer in co-cultures of keratinocytes and melanocytes. Immunofluorescence analysis using anti-tyrosinase and anti-human cytokeratin antibodies, phagocytic assays using fluorescent latex beads, and ultrastructural analysis indicated that KGF is able to induce melanosome transfer acting only on the recipient keratinocytes and as a consequence of a general role of KGF in the promotion of the phagocytic process. Inhibition of proteinase-activated receptor-2, to block the Rho-dependent phagocytic pathway, or of the Src family tyrosine kinases, to inhibit the Rac-dependent pathway, showed that KGF promotes phagocytosis through both mechanisms. Increased expression of the KGF receptor (KGFR) on the keratinocytes by transfection led to increased phagocytosis of latex beads following KGF treatment, suggesting that the KGF effect is directly mediated by KGFR expression and activation. Moreover, confocal microscopic analysis revealed that KGFR localize in phagosomes during KGF-induced phagocytosis, suggesting a direct role of the receptor in regulating both the early steps of uptake and the intracellular traffic of the phagosomes.

Many attempts have been made to obtain safe and effective analgesia in newborns. Oral glucose-water has been found to have analgesic properties in neonates. We investigated whether other sensory stimulation added to oral glucose provided more effective analgesia than oral glucose alone. In a randomized prospective double-blind trial, we studied 120 term newborns during heel prick. The babies were divided randomly into six groups of 20, and each group was treated with a different procedure during heel prick: A) control; B) 1 mL 33% oral glucose given 2 min before the heel prick; C) sucking; D) 1 mL 33% oral glucose plus sucking; E) multisensory stimulation including 1 mL 33% oral glucose (sensorial saturation); F) multisensory stimulation without oral glucose. Sensorial saturation consisted in massage, voice, eye contact, and perfume smelling during heel prick. Each heel prick was filmed and assigned a point score according to the Douleur Aiguë du Nouveau-né (DAN) neonatal acute pain scale. Camera recording began 30 s before the heel prick, so it was impossible for the scorers to distinguish procedure A (control) from B (glucose given 2 min before), C (sucking water) from D (sucking glucose), and E (multisensory stimulation and glucose) from F (multisensory stimulation and water) from the video. Procedure E (multisensory stimulation and glucose) was found to be the most effective procedure, and the analgesia was even more effective than that produced by procedure D (sucking glucose). We conclude that sensorial saturation is an effective analgesic technique that potentiates the analgesic effect of oral sugar. It can be used for minor painful procedures on newborns. Newborns feel pain (1, 2). Repeated painful stimuli lower their pain threshold (3, 4) by overstimulation of NMDA receptors, which may lead to excitotoxic brain damage (5). Until a few years ago, it was claimed that the word pain was inappropriate for newborns, as pain is a subjective experience that newborns, because of their age, cannot have (6). Until the 1980s, analgesics were rarely administered to newborns even in the case of surgery (7). Now we know that anesthesia reduces brain damage due to hypoxemia, hypertension, tachycardia, variations in heart rate, and increased intracranial pressure (8, 9), all of which are particularly dangerous because of immature cerebral vasoregulation in the premature (10).The number of painful stimuli needs to be kept to a minimum, and every effort should be made to render them less painful. Guidelines for neonatal analgesia have been suggested (11-15), especially for the most routine type of pain, blood sampling, which is usually performed by heel prick. To avoid the drawbacks of general and local analgesics (16 -21), types of nonpharmacologic analgesia have been proposed, including nonnutritional sucking and instillation of glucose or other sweet liquids on the newborn's tongue (22). The analgesic effect of glucose is thought to stimulate an increase in plasma concentrations of ␤-endorphin (23-27) by a preabsorptiv...

PERSPECTIVEhomology in the active site. These data would point a medicinal chemist to expect that it should be relatively easy to gain selectivity between CPT2 and CPT1 inhibitors, while it might prove to be a daunting task for a competitive inhibitor to differentiate CPT1A from CPT1B. An alternative option is to achieve tissue selective distribution of an unspecific inhibitor via permeability and pharmacodynamic properties.The homology between human, rat, and mouse enzymes, on the other hand, is rather high (see Table 3b), and species differences at the in vitro level are not likely to be significant (the rate of FAO in different species and the level of CPT activity control can be considerably different). 22 CPT1 is subject to multiple controlling factors. Expression level and basal activity of CPT1 depend on fasting state, 23 exercise, 24 type of diet, 25 exposure to cold, infections, metabolic disease, and enzyme inhibition. 9 Diabetes does not affect CPT1B activity 26 but increases CPT1A activity. 27 Malonyl-CoA, the first committed step of fatty acid synthesis, whose concentration is in turn highly regulated by multiple mechanisms, is a potent inhibitor of CPT1B (∼0.03 μM IC 50 for the rat enzyme) and a less potent inhibitor (by about 2 orders of magnitude) of CPT1A. 28,29 The sensitivity of CPT1A to allosteric inhibition by malonyl-CoA varies under different physiological conditions, amplifying the effect of the cellular malonyl-CoA concentration. In particular, declining malonyl-CoA concentrations reduce the sensitivity of the enzyme to allosteric inhibition (and vice versa). Mitochondrial outer membrane composition also affects sensitivity of CPT1A to malonyl-CoA. Studies regarding the binding of malonyl-CoA and other active-site directed inhibitors have shown that there are two separate binding sites for malonyl-CoA: a high-affinity binding site located on the cytoplasmic side of the protein and a second low-affinity site that corresponds to the catalytic site and where also CoA exerts a product-inhibition action. 30 It is postulated that one of these binding sites is a contact interaction between the N-and C-terminal segments of the protein, which explains the sensitivity to membrane fluidity as well as the loss of sensitivity to malonyl-CoA if the N-terminal portion of the protein is deleted (which also leads to loss of function) or by specific single point mutations in this region. The N-terminal domain is also responsible for the targeting of the enzyme to the outer mitochondrial membrane. 31 There is evidence that CPT activity is controlled by phosphorylation 32 and nitration, 33 although the hypothesis has been advanced that CPT is constitutively phosphorylated. 28

We present a series of small molecule drug discovery case studies where computational methods were prospectively employed to impact Roche research projects, with the aim of highlighting those methods that provide real added value. Our brief accounts encompass a broad range of methods and techniques applied to a variety of enzymes and receptors. Most of these are based on judicious application of knowledge about molecular conformations and interactions: filling of lipophilic pockets to gain affinity or selectivity, addition of polar substituents, scaffold hopping, transfer of SAR, conformation analysis, and molecular overlays. A case study of sequence-driven focused screening is presented to illustrate how appropriate preprocessing of information enables effective exploitation of prior knowledge. We conclude that qualitative statements enabling chemists to focus on promising regions of chemical space are often more impactful than quantitative prediction.

Adipose-derived stem cells (ASCs) represent a promising tool for soft tissue engineering as well as for clinical treatment of inflammatory and autoimmune pathologies. The well-characterized multi-differentiation potential and self-renewal properties of ASCs are coupled with their immunomodulatory ability in providing therapeutic efficacy. Yet, their impact in immune or inflammatory disorders might rely both on cell contactdependent mechanisms and paracrine effects, resulting in the release of various soluble factors that regulate immune cells functions. Despite the widespread use of ASCs in clinical trials addressing several pathologies, the pathophysiological mechanisms at the basis of their clinical use have been not yet fully investigated. In particular, a thorough analysis of ASC immunomodulatory potential is mandatory. Here we explore such molecular mechanisms involved in ASC immunomodulatory properties, emphasizing the relevance of the milieu composition. We review the potential clinical use of ASC secretome as a mediator for immunomodulation, with a focus on in vitro and in vivo environmental conditions affecting clinical outcome. We describe some potential strategies for optimization of ASCs immunomodulatory capacity in clinical settings, which act either on adult stem cells gene expression and local microenvironment. Finally, we discuss the limitations of both allogeneic and autologous ASC use, highlighting the issues to be fixed in order to significantly improve the efficacy of ASC-based cell therapy.