Both the ␣ and ␤␥ subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) communicate signals from receptors to effectors. G␤␥ subunits can regulate a diverse array of effectors, including ion channels and enzymes. G␣ subunits bound to guanine diphosphate (G␣-GDP) inhibit signal transduction through G␤␥ subunits, suggesting a common interface on G␤␥ subunits for G␣ binding and effector interaction. The molecular basis for interaction of G␤␥ with effectors was characterized by mutational analysis of G␤ residues that make contact with G␣-GDP. Analysis of the ability of these mutants to regulate the activity of calcium and potassium channels, adenylyl cyclase 2, phospholipase C-␤2, and ␤-adrenergic receptor kinase revealed the G␤ residues required for activation of each effector and provides evidence for partially overlapping domains on G␤ for regulation of these effectors. This organization of interaction regions on G␤ for different effectors and G␣ explains why subunit dissociation is crucial for signal transmission through G␤␥ subunits.Upon receptor activation, G proteins dissociate into free G␣ and G␤␥ subunits that can activate various effectors (1). Effector proteins of the G␤␥ complex include phospholipases (2), adenylyl cyclases (3), ion channels (4), G protein-coupled receptor kinases (5) and phosphoinositide 3-kinases (6). Other potential G␤␥ effectors include dynamin I and the nonreceptor protein tyrosine kinases Btk and Tsk (7). GDP-bound G␣ subunits (G␣-GDP) can compete with G␤␥ effectors and deactivate G␤␥-dependent signaling, suggesting that G␤␥ may use a common binding surface for interaction with G␣ and with its diverse effectors. Two regions on G␤␥ that interact with G␣ have been defined by the crystal structures of heterotrimeric G␣␤␥ (8), the switch interface (G␤ residues 57, 59, 98, 99, 101, 117, 119, 143, 186, 228, and 332) and the NH 2 -terminal interface (G␤ residues 55, 78, 80 and 89). Each of these residues on retinal G␤ (G␤1) was substituted with alanine, and each G␤1 mutant was expressed with either G␥1 or G␥2, two isoforms of the G␥ subunit. All mutated G␤1␥1 dimers were folded properly, were post-translationally modified appropriately, and were expressed at similar amounts as in the wild type (9). The G␤␥ mutants were tested for their ability to assemble into heterotrimers with G␣, to be activated by rhodopsin, and to interact with G␤␥ downstream signaling partners: ␤-adrenergic receptor kinase (␤ARK), phospholipase C-␤2 (PLC-␤2), adenylyl cyclase 2 (AC2), muscarinic potassium channel (GIRK1/GIRK4), and the calcium channel ␣1B subunit (CC␣1B).To determine whether purified G␤1H 6 ␥1 mutants could form heterotrimers, we measured the ability of the G␤␥ mutants to facilitate pertussis toxin-catalyzed adenosine diphosphate (ADP) ribosylation of transducin G␣-GDP (Gt␣) (10). All mutants could support some level of ADP ribosylation, although G␤ mutants Ile 80 3 Ala 80 (I80A), K89A, L117A, and W332A (11) showed reduced ability to form heterotrimers (Fig. 1A).Because G␤␥ ...

The type and amount of melanin synthesized by the melanocyte, and its distribution pattern in the surrounding keratinocytes, determines the actual color of the skin. Melanin forms through a series of oxidative reactions involving the amino acid tyrosine in the presence of the enzyme tyrosinase. Tyrosinase catalyses three different reactions in the biosynthetic pathway of melanin in melanocytes: the hydroxylation of tyrosine to l-DOPA and the oxidation of l-DOPA to dopaquinone; furthermore, in humans, dopaquinone is converted by a series of complex reactions to melanin. Among the skin-lightening and depigmenting agents, magnesium-l-ascorbyl-2-phosphate (MAP), hydroxyanisole, N-acetyl-4-S-cysteaminylphenol, arbutin (hydroquinone-beta-d-glucopyranoside) and hydroquinone (HQ) are the most widely prescribed worldwide. However, with reports of potential mutagenicity and epidemics of ochronosis, there has been an increasing impetus to find alternative herbal and pharmaceutical depigmenting agents. A review of the literature reveals that numerous other depigmenting or skin-lightening agents are either in use or in investigational stages. Some of these, such as kojic, glycolic and azelaic acids, are well known to most dermatologists. Others have been discovered and reported in the literature more recently. Several depigmentation and lightening agents are discussed, including their historical background, biochemical characteristics, type of inhibition and activators from various sources. In addition, the clinical importance of mushroom tyrosinase as a recent prospect is discussed in this paper.

Tyrosinase is a copper-containing enzyme, which is widely distributed in microorganisms, animals and plants and is a key enzyme in melanin biosynthesis, involved in determining the color of mammalian skin and hair. In addition, unfavorable enzymatic browning of plant-derived foods by tyrosinase causes a decrease in nutritional quality and economic loss of food products. The inadequacy of current conventional methods to prevent tyrosinase action encourages researchers to seek new potent tyrosinase inhibitors for food and cosmetics. This article presents a study on the importance of tyrosinase, biochemical characteristics, type of inhibitions, activators from various natural sources with its clinical and industrial importance in recent prospects is discussed in this paper.

The G protein transducin has been an often-used model for biochemical, structural, and mechanistic studies of G protein function. Experimental studies have been limited, however, by the inability to express quantities of mutants in heterologous systems with ease. In this study we have made a series of G alpha t/G alpha i1 chimeras differing at as few as 11 positions from native G alpha t. Ten chimeras are properly folded, contain GDP, can assume an A1F4(-)-induced activated conformation, and interact with beta gamma t and light-activated rhodopsin. They differ dramatically in their affinity for GDP, from Gi-like (initial rates 225 mumol/mol s) to Gt-like (initial rates 4.9 mumol/mol s). We have used these chimeras to define contact sites on G alpha t with the effector enzyme cGMP phosphodiesterase. G alpha t GTP but not G alpha t GDP activates it by removing the phosphodiesterase (PDE) gamma inhibitory subunit. In solution, G alpha t GTP interacts with PDE gamma (Kd 12 nM), while G alpha t GDP binds PDE gamma more weakly (Kd 0.88 microM). The interaction of G alpha i GDP with PDE gamma is undetectable, but G alpha i GDP-A1F4- interacts weakly with PDE gamma (Kd 2.4 microM). Using defined G alpha t/G alpha i chimeras, we have individuated the regions on G alpha t most important for interaction with PDE gamma in the basal and activated states. The G alpha t sequence encompassing alpha helix 3 and the alpha 3/beta 5 loop contributes most binding energy to interaction with PDE gamma. Another composite P gamma interaction site is the conserved switch, through which the GTP-bound G alpha t as well as G alpha i1 interact with P gamma. Competition studies between PDE gamma and truncated regions of PDE gamma provide evidence for the point-to-point interactions between the two proteins. The amino-terminal 1-45 segment containing the central polycationic region binds to G alpha t's alpha 3 helix and alpha 3/beta 5 loop, while the COOH-terminal region of P gamma, 63-87, binds in concert to the conserved switch regions. The first interaction provides specific interaction with both the GDP- and GTP-liganded G alpha t, while the second one is conserved between G alpha t and G alpha i1 and dependent on the activated conformation.

The recognition between G protein and cognate receptor plays a key role in specific cellular responses to environmental stimuli. Here we explore specificity in receptor-G protein coupling by taking advantage of the ability of the 5-hydroxytryptamine 1B (5-HT 1B ) receptor to discriminate between G protein heterotrimers containing G␣ i1 or G␣ t . G i1 can interact with the 5-HT 1B receptor and stabilize a high affinity agonist binding state of this receptor, but G t cannot. A series of G␣ t /G␣ i1 chimeric proteins have been generated in Escherichia coli, and their functional integrity has been reported previously (Skiba, N. P., Bae, H., and Hamm, H. E. (1996) J. Biol. Chem. 271, 413-424). We have tested the functional coupling abilities of the G␣ t /G␣ i1 chimeras to 5-HT 1B receptors using high affinity agonist binding and receptor-stimulated guanosine 5-3-O-(thio)triphosphate (GTP␥S) binding. In the presence of ␤␥ subunits, amino acid residues 299 -318 of G␣ i1 increase agonist binding to the 5-HT 1B receptor and receptor stimulation of GTP␥S binding. Moreover, G␣ i1 containing only G␣ t amino acid sequences from this region does not show any coupling ability to 5-HT 1B receptors. Our studies suggest that the ␣4 helix and ␣4-␤6 loop region of G␣s are an important region for specific recognition between receptors and G i family members.The heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) mediate signaling from a large number of diverse heptahelical cell surface receptors to a variety of intracellular effectors. These pathways control numerous essential functions in all tissues and are ubiquitous throughout eukaryotes (1-3). A large body of work investigating the mechanisms underlying receptor-G protein interactions now exists. The early view that signaling selectivity would manifest itself on the basis of specific protein interactions allowing a receptor to couple with a unique G protein to modulate a single effector is no longer tenable with the accumulating evidence of a network of interactions that converge and diverge at multiple levels. Even in the earliest receptor-G protein reconstitution studies using phospholipid vesicles, it was clear that, while there were large differences in the efficiencies of coupling among the major families of G proteins, receptors were capable of activating multiple G proteins from distinct families (4, 5).Elucidation of the crystal structures of ␣ subunits in both active (6, 7) and inactive conformations (8), an isolated ␤␥ subunit (9) and the ␣␤␥ heterotrimer (10, 11), has begun to define a mechanistic basis for data from mutagenesis, chimera, and peptide studies defining functional domains on G protein subunits (12)(13)(14)(15)(16)(17). A variety of studies have implicated the C terminus of ␣ subunits in mediating receptor-G protein selectivity (13-15). Synthetic peptides from the C terminus of ␣ t (amino acids 340 -350) have been shown to stabilize the active conformation of metarhodopsin II (17) while alanine scanning mutagenesis of the same regi...

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Current results demonstrated distinctive personality traits associated with Sasang types using reproducible psychometric and anthropometric instruments. With further study, the Sasang typology could serve as a scientific tool for individualized and integrative medicine.

Abstract:Inflammation is a pervasive phenomenon triggered by the innate and adaptive immune systems to maintain homeostasis. The phenomenon normally leads to recovery from infection and healing, but when not properly phased, inflammation may cause immune disorders. Bee venom is a toxin that bees use for their protection from enemies. However, for centuries it has been used in the Orient as an anti-inflammatory medicine for the treatment of chronic inflammatory diseases. Bee venom and its major component, melittin, are potential means of reducing excessive immune responses and provide new alternatives for the control of inflammatory diseases. Recent experimental studies show that the biological functions of melittin could be applied for therapeutic use in vitro and in vivo. Reports verifying the therapeutic effects of melittin are accumulating in the literature, but the cellular mechanism(s) of the anti-inflammatory effects of melittin are not fully elucidated. In the present study, we review the current knowledge on the therapeutic effects of melittin and its detailed mechanisms of action against several inflammatory diseases including skin inflammation, neuroinflammation, atherosclerosis, arthritis and liver inflammation, its adverse effects as well as future prospects regarding the use of melittin.