text: Tony Hawk's Pro Skater 3 - Review A+ A- Email Out of all the many, many 'Tony Hawk' games out there on the market this has always been the one that stood out to me alongside 'Tony Hawk Pro Skater 4'. Looking back at it, it's easy to see why with its seriously fun level design, highly customizable features and challenging but achievable goals it was a game that could steal hours of my time. On the surface this may appear to be a simple skating game but as you delve deeper you'll realize that it's jam packed with secrets, unlockables, challenges and memorable moments that belay its unique structure. Developer: Neversoft Genre: Sports Release date: October 28th 2001 Platforms: PlayStation, PlayStation 2, Xbox, Nintendo GameCube, Nintendo 64, Microsoft Windows, ESRB: E - Everyone Although this game is over fifteen years old it was, in its time, a groundbreaking addition to the sports game market by providing the player with a massive amount of freedom in its open world level design whilst setting up quirky but challenging objectives. These days it looks a little rough around the edges but it is in no way less fun than it ever was and even without the modern day perks of things like, being able to get off the board or a detailed storyline, it's still a fantastic game. This 'Tony Hawk' game has various modes that you may play including career mode, single session, free skate and 2 player co-op games. There are also a couple of customization modes including park editor, where you create your own park that can be used for free skate or 2 player games and there's also a create a skater mode too. I found the career mode to be the most entertaining as rather than it consisting of having your skater achieve world fame, like many of the later Tony Hawk's games, it's simply a set of levels that all have some objectives you need to try and complete in a set time of around about 2 to 3 minutes. Doesn't sound like a lot of time does it? Well, it isn't, and it'll take you many replays of each level in order to complete the goals that are set. Each level has its own unique goals but generally they follow a guideline of get a high score, get a pro score, grab the secret tape which is hidden somewhere awkward in the level, do 5 of something specific, grab the letters of the word 'SKATE' and then complete a few special ones for that level. Special objectives include things like 'grind the molten bucket' in the foundry level which involves triggering a bucket to descend from the ceiling, allowing it to pour some lava into a pit then somehow managing to jump and grind along the bucket itself. Hidden within levels are also collectibles like the odd new deck that you can give a custom skater and that special tape which is a clip of real life footage. Since these unlockables never really interested me it wasn't really the allure of a reward that drove me to play the game so intensely and the only way I can describe it really is that the game is just a great deal of fun to play. The controls feel solid, the environments are interactive and there's always the chance of stumbling upon a completely new area since the game likes to hide things from you and reward you for exploring the map. The levels themselves are incredibly varied and you'll get to visit locations like snowy Canada explore the canopy of an enormous tree or Surburbia where you could explore the secrets of a haunted mansion and smash up some pumpkins. All of the levels have some secret location to find that can be unlocked by smashing through a certain area, triggering a bit of the environment to change or just by reaching somewhere really difficult to get to. As is normal for the Tony Hawk games the tone is very comedic and not at all realistic so you'll be able to do all sorts of bizarre things and skate and trick your way off of almost anything. There are twelve levels for all versions of the game except the older consoles including the PlayStation and the Nintendo 64 which only have nine levels, one of which is exclusive to them. I found the idea of this kind of irritating because rather than encouraging people to do something extra to play another level it's more of a way of excluding people from the full content. It's not as though we're going to buy another console just to play the one exclusive level so what's the purpose of it? Who knows, but it is a minor niggle. On the plus side the contents of the levels are very dynamic with moving cars, that will knock you flying if you crash into one, and plenty of NPCs wandering about who will frequently make comments about you. Depending on what you're doing there are many parts of the environment that can be changed or destroyed, normally revolving around the objectives, and this results in a mini-cutscene so you can view the full glory of what you've done. In this sense there's a lot of cinematic quality about the game and it's not just a matter of you skating around and performing tricks as, although there's no real storyline, there's definitely a background dialogue going on that makes it something more than your typical sports game. The other modes of the game take place in the regular levels available to you in career mode but without a time constraint, allowing you to do things like search for gaps or secrets in the level. The single session mode is timed and is an opportunity to set a high score within one of the levels under the regular 2 minute timer. Really the only other game mode worth going into detail in is the multiplayer modes. Previously there was a network option for certain consoles but it's so old now they're not in use so instead I'll focus on the extremely enjoyable co-op mode. Within the multiplayer mode you can have up to three more players join in and play with you on any of the regular levels or one of your user created levels. You can pick from modes to play such as free skate, where you just mess around and get high scores, graffiti where you compete against one another and against the clock to capture as much territory as possible by performing a trick on it. Once that object is coloured the other player will need to perform a trick with a higher score than the previous player's object score in order to claim it for themselves. Another mode, called HORSE, allows you to try and continually beat the other player's combo score or else get a letter of the word HORSE, or a custom word chosen by you, whereby if you spell HORSE then you lose the game. Lastly there's the game Slap! where you quite literally skate around and try and hit one another whereby the person with the most slaps wins annnd finally there's King of the Hill where you need to grab a floating crown and then hold onto it for the longest time possible. If another player rams into the king then they get the crown instead. It's all a bit ridiculous but extremely enjoyable, especially when you're trying ot play this on a map customized by you to have an entire floor of lava and a whole bunch of grind rails everywhere bar the odd safe area. Heh, good times. It's got to be said that the feel of the game is spot on especially when it comes to the balancing mechanic when performing moves like grinds, manuals and lip tricks. The variety of ticks you can perform is also enormous and there are lots of comical ones that tend to revolve around where you are on the map and what you've just done. There are essentially five categories of tricks; grinds, lips, flips, grabs and manuals but all of these can be combined and all of these have multiple variations of themselves. By stringing together lots of different tricks you can gradually create an enormous combination and this is the first Tony Hawk game to allow players to use the revert, a trick that enables vert combos to be strung together by using a manual after landing in a quarterpipe. This move allows for much longer combos and thus much higher scores. Something I found surprising was that this is also the first game to introduce the balancing mechanic for lips and rails, this requires the player to manually adjust a bar in the middle of the balance bar in order to maintain their balance. If you slip and the bar goes too far in one direction you'll normally fall over though this depends on where you are at the time so, for example, if you're currently balancing on a lip and your bar slips too far to the bottom you'll simply roll off and back down the halfpipe rather than fall off your board. Although the game is getting on in years now, it was released in 2001 so let's be reasonable here, it looks great. The draw distance in particular is fantastic and the graphics themselves are excellent for this year although on the older consoles they are significantly worse. The voice acting is a little cheesy but since the tone of the game is a bit wacky it strangely fits in with the humour and since the rest of the audio is top notch I don't really mind. The soundtrack is traditionally very cool and very much a reflection of that era of grunge, rock and hip hop music with artists such as the Red Hot Chili Peppers, Motorhead and House of Pain adding in their own tracks. In terms of customizability of the game there's a fair bit you can tweak about with such as creating your own skater in what is now considered to be a fairly limited workshop but where you can pick their appearance and what kind of board they use. Lastly you can make your own skate park by placing objects and obstacles in a large empty area then saving it to free skate or compete in against a friend later on. This is one of my favourite Tony Hawk games and this is because it is simply an extremely fun game. It's quirky, interactive and challenging with great co-op modes and some really challenging moments, especially when you're trying to complete the career mode. The game is also very accessible and if you, say, struggle with the balance bars there are cheats for almost everything and they can simply be turned off. You can unlock hidden characters like Darth Maul and a flaming demoness as well as make things just that little bit more ridiculous by flicking on slow-mo mode or giant/tiny mode. All of the cheats in the game can be unlocked via playing the game legit also so if you're not a cheater then you can still mess around as nature intended. Overall, even though I'm not a fan of sports games, this is well worth playing and most definitely falls within the golden years of skateboarding games. The Good: Huge variety of gameplay modes Time limits on levels makes you value every moment of play Fantastic co-op modes Lots of unusual characters like Darth Maul and Wolverine to play as The create-a-park feature is great for multiplayer Surprisingly good graphics for a 2001 game Excellent soundtrack Large levels with secrets to find and unlock Solid controls that make skating an enjoyable experience Lots of humour, especially in its NPCs The Bad: The challenges for the career mode could be more varied from level to level Kind of annoying how the different platforms come with different levels Practically zero storyline Lacking in depth compared to more recent titles The Score: 8/10Final thoughts: "This is one of my favourite skating games of all time, it's a good mixture of casual messing around and hardcore completioninist gameplay."By: Rambling Fox Reviews Posted by Fox Rambles Labels: 2001, Co-op Games, Comedy Games, Nintendo 64 Games, Nintendo Gamecube Games, PlayStation 1 Games, PlayStation 2 Games, Retro Games, Skateboarding Games, Sports Games, Twitch controls, Unlockables, Xbox Games Share to: at 15:12


text: One of Karai's aides from the 2003 TV series he appears in the City at War episodes where he helps Karai fight the Turtles, Casey Jones, the Foot Clan, the Purple Dragons, and the Mafia. As well as the episodes Good Genes, Part 2 where Karai orders him and the Tall Karai aide to investigate a power outage. Neither of the aides had an official name, though Peter Laird suggested during developmental to call one "Yin" and the other "Yang".[1]

subset: commoncrawl


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- Review
- Open Access
The roles played by highly truncated splice variants of G protein-coupled receptors
- Helen Wise1_91Email author
© Wise; licensee BioMed Central Ltd. 2012
- Received: 11 April 2012
- Accepted: 29 July 2012
- Published: 1 September 2012
Alternative splicing of G protein-coupled receptor (GPCR) genes greatly increases the total number of receptor isoforms which may be expressed in a cell-dependent and time-dependent manner. This increased diversity of cell signaling options caused by the generation of splice variants is further enhanced by receptor dimerization. When alternative splicing generates highly truncated GPCRs with less than seven transmembrane (TM) domains, the predominant effect in vitro is that of a dominant-negative mutation associated with the retention of the wild-type receptor in the endoplasmic reticulum (ER). For constitutively active (agonist-independent) GPCRs, their attenuated expression on the cell surface, and consequent decreased basal activity due to the dominant-negative effect of truncated splice variants, has pathological consequences. Truncated splice variants may conversely offer protection from disease when expression of co-receptors for binding of infectious agents to cells is attenuated due to ER retention of the wild-type co-receptor. In this review, we will see that GPCRs retained in the ER can still be functionally active but also that highly truncated GPCRs may also be functionally active. Although rare, some truncated splice variants still bind ligand and activate cell signaling responses. More importantly, by forming heterodimers with full-length GPCRs, some truncated splice variants also provide opportunities to generate receptor complexes with unique pharmacological properties. So, instead of assuming that highly truncated GPCRs are associated with faulty transcription processes, it is time to reassess their potential benefit to the host organism.
- Alternative splicing
- Cell signaling
- G protein-coupled receptors
- Truncated splice variants
G protein-coupled receptors (GPCRs) constitute the most abundant gene family in most animal species [1] and are the target of at least 50% of marketed drugs [2]. The number of GPCRs varies widely among different species, with the elephant having almost three times as many genes as the 1265 found in humans [3]. The question then is, "does this matter?" GPCRs are not only receptors for light and for biologically active chemicals, but also respond to stimuli associated with smell and taste. Therefore, the higher number of GPCR genes in the elephant may just reflect its greater reliance on its sense of smell. However, simply counting the number of genes in a genome greatly underestimates the number of gene products actually produced because of a process known as alternative splicing. By overlooking the number and variety of GPCR splice variants, we may misinterpret the fine-tuning options for controling cell signaling activity in health and disease. Furthermore, it has been proposed that GPCR oligomerization also provides a way to increase the number of receptor entities with a limited number of genes [4, 5], and even apparently non-functional highly-truncated splice variants of GPCRs can generate functionally active GPCRs by heterodimerization [6, 7]. To add further to this complexity of GPCR signaling, even though individual cell types typically express more than one hundred different GPCRs, the most highly expressed GPCRs are not necessarily those which have been targeted therapeutically [3]. So even if splice variants are expressed less well than their wild-type counterparts, this does not automatically mean they are of little consequence. Because dysregulation of GPCR activity contributes to many different pathophysiological processes [8], we still need to carefully consider the specific role of each GPCR and its splice variants. Alternative splicing generates GPCRs with variations in specific structural domains, and in this review we will focus on the highly truncated splice variants which represent approximately 50% of all GPCR isoforms studied to date. We will show that generalizations assuming highly truncated GPCRs are non-functional may be an oversimplification, and we will discuss how GPCR dimerization between wild-type receptors and their splice variants has added a new level of regulation to the complex process of cell signaling. This review will also focus on some unique splice variants offering insights into the physiological role of these GPCR isoforms.
Alternative splicing of genes
GPCR trafficking – ER retention of highly truncated splice variants
GPCRs are seven-transmembrane (7TM) domain receptors (Figure 1) which are trafficked through the biosynthetic pathway to the cell surface in a tightly regulated mechanism with multiple steps and a stringent quality control system to ensure correct GPCR folding and targeting. Association of GPCRs with accessory proteins or chaperones are a key step for the forward trafficking through the endoplasmic reticulum (ER) and Golgi [15]. The life of GPCRs begins in the ER where they are synthesized, folded and assembled [16, 17]. During their migration to the cell surface, GPCRs undergo post-translational modifications to attain mature status. While knowledge of the structure-function relationship of GPCRs has been extended by recent high-resolution structural studies of β2-adrenoceptors [18] and muscarinic M2 receptors [19], little is known of features influencing overall stability of the highly truncated mutant GPCRs. In some cases, alternative splicing generates mutant transcripts which are simply too faulty to be expressed; splice variants of the human endothelin (ETB) receptor do not appear to be translated, or the products are quickly degraded, presumably because of their instability [20]. Nevertheless, the preferential production of this null function ETB by RNA editing/splicing has been proposed to underlie the etiology of Hirshsprung disease [20], so even the most unstable GPCRs can impact on overall physiology.
GPCR splice variants with altered or deleted transmembrane domains
Splicing consequence
General properties of splice variant
Truncated 6TM mutants.
No cell surface expression.
Calcitonin receptor (CT receptor)
Truncated 6TM mutant.
Rabbit CTRΔe13 is poorly expressed on the cell surface and fails to activate cell signaling.
Chemokine receptor 5 (CCR5)
Severely truncated mutants. Ccr5Δ32 is a 5TM mutant.
Natural mutants of human chemokine receptor CCR5 lacking the last 3 or 5 TMDs are non-functional. Ccr5Δ32 complexes with CCR5 and retains CCR5 in the ER, thus reducing cell surface expression.
Dopamine receptor (D3)
D3nf is a truncated 5TM mutant.
Human D3nf mutant has a punctuate perinuclear distribution and does not bind DA-ligands.
GABAB receptor (GABABR1)
GABABR1c has an additional 31 amino acids in TM5.
Gastric inhibitory polypeptide receptor (GIP )
Truncated 4TM mutant.
This inactive mutant receptor inhibited GIP signaling and decreased cell surface expression by retaining WT receptor in the ER.
Ghrelin receptor (or GHS-R1a)
GHS-R1b is a truncated 5TM mutant.
GHS-R1b is a non-signaling splice variant of GHS-R1a.
Gonadotropin-releasing hormone receptor (GnRH receptor)
Truncated 5TM mutant.
5TM variant of human GnRH receptor shows decreased cell surface expression, no ligand binding and no signal transduction.
Growth hormone-releasing hormone receptor (GHRH-R)
Truncated 5TM mutant.
The mutant GHRH-R cannot transduce GHRH signals.
Histamine receptor (H3)
6TM-rH3R is a truncated 6TM mutant.
Several splice variants of rat H3R do not bind agonist, have an intracellular localization and co-expression with WT receptor decreases cell surface expression and functional responses.
Histamine receptor (H4)
hH4R(302) lacks 88 amino acids from TMD2 to TMD4. hH4R(67) is a ~ 1.5TM truncated mutant.
These human H4R splice variants were localised predominantly intracellularly when expressed in recombinant cells. No ligand binding or cell signaling detected.
Leukotriene B4 receptor (BLT1)
LTB4R-AS1 lacks TMD2 and part of ECL1 (39 amino acid deletion).
Both isoforms of human BLT1 (LTB4R) are expressed in human airway smooth muscle cells.
LTB4R-AS2 is the 3TMD to C-terminus (lacks 100 amino acids).
Luteinizing hormone receptor (LH receptor)
Truncated 5TM mutant.
The 5TM mutant of human LH receptor binds ligand (limited) but has no signaling activity.
μ-opioid receptor (μ-OR)
Truncated 6TM mutant.
A 6TM μ-OR variant in mice identified ligands lacking the traditional side effects of classical opiates but maintaining analgesic property.
Motilin receptor
GPR38-B is a truncated 5TM mutant.
Neurokinin 2 receptor (NK2)
An ICL2-TM4 deletion mutant.
NK2β splice variant is poorly expressed on the cell membrane and is non-signaling.
Neuropeptide Y receptor (Y1)
A truncated 5TM mutant.
The putative hY1-related 5TM accessory protein encoded by the non-spliced hY1 mRNA is not involved in facilitating hY1 production.
Neurotensin receptor (NTS2)
Truncated 5TM mutant with long tail.
Rat vNTS2 is functionally active and can heterodimerize with NTS2.
Nociceptin receptor (NOP)
Truncated 4TM mutant.
The rat truncated NOP receptor is localised to cell membranes but is non-functional.
Prostaglandin F2α receptor (FP)
A 6TM truncated mutant (FPs)
hFPS is functionally inactive and highly expressed in the perinuclear region.
A truncated mutant (PTGFR-v2).
No distinct functional role identified.
Somatostatin receptor (sst5)
Human sst5TMD5 is a 5TM truncated mutant.
Identified novel truncated but functional human sst5-variants; present in normal and tumoral tissues.
Human SST5TMD4 is a 4TM truncated mutant.
Murine sst5TMD4 is a 4TM truncated mutant.
Three murine variants were functional to mediate ligand-selective-induced variations in Ca2+ and cAMP despite being truncated and displayed a preferential intracellular distribution.
Murine sst5TM2 is a 2TM truncated mutant.
Murine and rat sst5TM1 has just TM1.
Vasopressin receptor, subtype 2 (V2)
V2b is a truncated 6TM mutant.
V2b is retained in the ER where its C-terminus can be either intracellular or extracellular.
For the GnRH receptor, a single change in net charge is sufficient to tip the balance in favour of the ER and diminish GnRH receptor available at the plasma membrane [52]. Conn et al. (2006) [22] have proposed that the apparent inefficiency of the GnRH receptor must have evolved under strong and convergent evolutionary pressure, suggesting there must be a strong advantage to generating an inefficiently produced receptor which is highly susceptible to cause a mutational disease. Whether such unexpected evolutionary pressure also exists for the ER-retained highly truncated GPCRs is unknown.
GPCR dimerization and its influence on GPCR trafficking
It is becoming apparent that many GPCRs form homo and/or hetero-dimers or higher order oligomers and that dimerization could both positively and negatively regulate GPCR cell surface targeting [53]. But it is important to realize that some functional consequences that are proposed to originate from heteromeric receptor interactions may also be observed due to intracellular crosstalk between signaling pathways of non-associated GPCRs [54]. Heterodimerization between different GPCR subtypes can significantly modify functional characteristics of the individual protomers, included subcellular localization, ligand binding co-operativity and proximal signaling [55].
That homodimerization was a prerequisite for cell surface targeting was first identified with the β2-adrenoceptor [21]. A few examples of other GPCRs with constitutive dimers/oligomers which form during biosynthesis are: α1D-adrenoceptors (in complex with β2-adrenoceptors) [56]; GABAB receptors [57]; ghrelin receptors [58]; gonadotrophin hormone (LH/hCG and FSH) receptors [59, 60]; neurotensin NTS1 and NTS2 receptors [61]; oxytocin receptors [62]; and vasopressin V1a and V2 receptors [62]. Collectively, most data suggests that receptor oligomers are preassembled in the ER and 'walk hand-in-hand' to the cell surface [54].
The impact of GPCR heterodimerization on cell surface targeting is perhaps best exemplified by GABAB receptors [63]. The GABAB receptor (GABABR1) did not couple effectively to expected signaling pathways until co-expressed with GABABR2 which allowed GABAB1R to escape from the ER [64]. Expression of the GABABR1 subunit on the cell surface was prevented through a C-terminal retention motif which needed to be masked by heterodimerization with GABABR2 [64]. In contrast, the heterodimerization of α1A- and α1D-adrenoceptors primarily involves the hydrophobic core of these receptors as deletion of the C-terminal domains did not affect cell surface expression [65]. While there is evidence for specific heterodimerization between wild-type GPCRs and highly truncated isoforms in the ER [58], the structural motifs responsible for heterodimerization with truncated splice variants and subsequent ER retention are unknown. It is currently possible to rescue GPCRs retained in the ER using chemical or pharmacological chaperones [66], and this presents an interesting approach to overcome the dominant-negative effect of the highly truncated GPCR splice variants.
Evidence of heterodimerization of full length GPCRs in vivo has been harder to obtain, [67], but examples are available associated with pathophysiological conditions: angiotensin-II (AT1) – bradykinin receptor complexes in preeclampsia [68]; the μ-opioid receptor and α2A-adrenoceptor complex in depression and opioid addiction [69]; the adenosine A2A – dopamine D2 receptor complex in Parkinson's disease [70]; the κ-δ-opioid receptor complex in analgesia and drug abuse [71] and the metabotropic glutamate (mGlu2) – 5-hydroxytryptamine (5-HT2A) receptor complex in schizophrenia [72, 73]. As highly truncated splice variants are relatively hard to detect in vivo, definite proof of the existence of their heterodimerization with full length GPCRs is harder to obtain (see next section).
To address the issue of whether or not truncated GPCRs are functionally active, we need to consider if ER-retained GPCRs are capable of signaling activity. There is a growing body of evidence supporting the hypothesis that endocytosed receptors can activate specific signal transduction pathways [74]. But what about signaling from the ER before trafficking of receptors to the cell surface? Studies with β2-adrenoceptor have shown that during its trafficking through the ER/Golgi, this GPCR is already pre-associated with its G proteins and effector enzyme (adenylyl cyclase 2) in a signaling complex [75]. Another example of a GPCR with activity in the ER is the ghrelin receptor. Constitutive activation of extracellular signal-regulated kinases (ERK1/2) can be detected in the ER of HEK293 cells expressing ghrelin receptors [58]. Both observations support the idea that GPCR signaling can be activated in the ER, but whether or not GPCR signaling in the ER is fundamentally different from GPCR signaling at the cell surface remains to be determined.
For non-constitutively active GPCRs, there is the issue of access of agonist to the receptor when it is retained in the ER. Nuclear metabotropic glutamate (mGlu5) receptors are orientated with their ligand binding domain inside the nucleoplasmic reticulum, and an active transport process is used so that glutamate can access their binding sites in this intracellular compartment [76]. As the ER is contiguous with the nuclear membrane [77], it is possible that similar systems exist to facilitate cell signaling of other GPCRs from this intracellular location. For GPCRs such as the thyroid-stimulating hormone (TSH) receptor, activation of adenylyl cyclase in different subcellular compartments regulates different cellular functions [78]. Ultimately, one should not necessarily assume that ER-retained GPCRs and their splice variants will be non-signaling, or that they will serve the same function as receptors on the cell surface.
Splice variants of GPCRs - Introduction
Previously, it was assumed that splice variants of ligand-gated channels were common, whereas each GPCR was far more likely to be a single product of its gene [79]. Thus, initial estimates suggested that approximately 90% of mammalian GPCRs were intronless in their open reading frame, which compares with approximately 35% for other cell surface receptors [80]. Access to additional databases and bioinformatics studies now indicates that perhaps only 50% of mammalian GPCR genes are intronless and do not require post-transcriptional processing [81]. The relative absence of introns in human GPGRs could indicate that most GPCR genes were derived from a single intronless common progenitor relatively recently in evolutionary history [80]. Because intronless genes do not require post-transcriptional splicing, we might expect them to be transcribed more efficiently [80], but this in turn would decrease diversity. After all, GPCR genes that do possess introns can undergo alternative splicing, generating GPCR subtype isoforms that may differ in their pharmacological, signaling and regulatory properties. Splice variants of GPCRs were often dismissed as the consequence of 'leaky transcription' and hence deemed physiologically irrelevant [82]. Despite this, we see that splice variants of GPCRs are not uncommon, and that formation of heterodimers and/or retention of functionally-active GPCRs in different subcellular compartments greatly increases the complexity of GPCR signaling.
The largest group of splice variants per GPCR relates to the C-terminus [79, 82] but distinct differences can exist between human and rat/mouse orthologs such as the EP3 receptor [83], making it essential to understand the relationship between human and non-human GPCRs before inferring mechanisms based on non-human receptor studies. When genomic libraries were first prepared, the possibility of alternative gene splicing was generally ignored [79], with the first clear evidence in 1989 pertaining to the rat dopamine D2 receptor [84]. GPCR splice variants often show differential distribution among many tissues and brain regions, consistent with cell-specific control of transcription and splicing [82], and alternative versions of a gene transcript might be necessary for different tissue types or at different stages of life [80]. Thus, alternative splicing serves as a molecular tool to introduce more diversity into gene expression and this may have been generated as a more economical alternative to gene duplication during evolution [82].
Given that GPCRs can exist as dimers or oligomers, we might expect to observe dominant-negative effects in some heterozygous individuals, which could relate to defective routing of the complex formed between the wild-type and mutated receptors to the plasma membrane [85]. Some clinical evidence described in obese patients with mutations in melanocortin (MC4) receptor supports this concept, but, with a few exceptions, expression of the disease in heterozygous individuals is usually mild or absent [86].
Alternative splicing of the GPCR superfamily in human airway smooth muscle has recently been demonstrated to diversify the complement of receptors [41]. Out of the 434 GPCRs detected in airway smooth muscle, 192 GPCRs had, on average, five different expressed receptor isoforms. There was no apparent relationship between 'wild-type' expression levels and the occurrence of a particular splicing event. Of note in this study was the relatively low expression levels of GPCRs such as the M3-muscarinic receptor, β2-adrenoceptor, and receptors for leukotrienes and prostanoids which are already targeted therapeutically. Therefore, a low expression level of GPCRs does not necessarily equate to functional insignificance.
The dominant-negative effect of GPCR splice variants with altered or deleted transmembrane domains, generating highly truncated mutant receptors
For GPCRs with relatively large N-terminal ectodomains, alternative splicing can generate truncated receptors lacking the N-terminal domain. Cleaving the N-terminal domain from the remaining TM domains generates a more specific dominant-negative effect due to the released N-terminal domain (Figure 2). Hence, soluble non-membrane anchored 'receptors' are seen for the corticotrophin-releasing factor receptor (CRF2) [90], metabotropic glutamate (mGlu6) receptor [91] and the relaxin receptor (RXFP1) [92]. These secreted proteins are proposed to act as biological modifiers of endogenous ligand-stimulated activity by mopping up endogenous ligand and preventing activation of the respective wild-type receptors.
Association of truncated GPCRs with pathophysiological conditions
Although there are some exceptions (see sst5 receptors above), splice variants resulting in truncated GPCRs of just 4 – 5 TM domains tends to generate functionally inactive mutant receptors. In Table 1 we have 22 examples of truncated GPCRs and the majority have no cell surface expression and no cell signaling activity. These mutant GPCRs are retained in an intracellular compartment, typically the ER and frequently function as dominant-negative mutants of the wild-type receptors. Retaining the wild-type receptors in the ER can have significant functional consequences, dependent on the constitutive role of the wild-type GPCR and/or its role in mediating responses to pathogenic organisms. For example, the chemokine receptor 5 (CCR5) functions as a co-receptor for HIV-1 infection, thus mutations allow for a protective role against HIV infection [30]. The 5TM ccr5Δ32 splice variant complexes with and retains CCR5 in the ER, so the ccr5Δ32/ccr5Δ32 genotype has been linked with a phenotype that is "highly" protected from HIV-1 infection, whereas the CCR5/ccr5Δ32 genotype confers only "relative" protection [31]. As seen with CCR5 and its 5TM splice variant, formation of specific heterodimers between truncated GPCRs and their associated wild-type receptors has been identified with the dopamine D3 receptor [32, 33], ghrelin receptor mutant polypeptide (GHS-R1b) [36] and histamine H4 receptor [40], but the association with a specific pathology remains speculative. The intracellular localization pattern of the other 5TM mutants listed in Table 1 strongly suggests they too form heterodimers with corresponding wild-type GPCRs resulting in decreased cell surface expression of these receptors. Unfortunately, the absence of antibodies capable of distinguishing wild-type and truncated GPCRs in vivo continues to hamper developments in this area.
While intracellular retention of wild-type GPCRs can arise by heterodimerization with 5TM mutants, there are other means to ultimately decrease expression of wild-type GPCRs. In schizophrenia, there is a decrease in dopamine D3 receptor mRNA but no change in 5TM D3nf mutant [93]. The enhanced D3nf-specific splicing of D3 pre-mRNA in schizophrenia may lead to this decreased expression of D3 mRNA, and thus a decrease in D3 receptor protein [94]. To generate D3nf mRNA by alternative splicing requires a rare splice event capable of recognising just 98 nucleotides as an alternative intron within a larger 2675 nucleotide exon [94]. Despite its rarity, this splicing event clearly occurs.
The differential expression of truncated splice variants is perhaps the norm [40, 47, 49, 95, 96], but there are always exceptions. The somatostatin (sst5) receptor splice variants showed no marked differential expression, but they were differentially regulated by changes in hormone/metabolic environment in a tissue- and ligand-dependent manner [50]. An altered hormone/metabolic environment resulting from a high-fat diet changes the ratio of expression of gastric inhibitory polypeptide (GIP) receptor and its 4TM mutant in mice [35]. The relative reduction of truncated GIP receptor expression may be involved in the hypersensitivity of GIP and hyperinsulinemia in these obese mice. Similarly, the expression of luteinizing hormone (LH) receptor and its 5TM truncated splice variant (LHd) in the corpus luteum varies at different stages of the ovarian cycle [96]. This profile of independent regulation for sst5, LH and GIP and their splice variants in response to different physiological conditions contrasts with the prostaglandin F2α (FP) receptor splice variants. FP receptor splice variants are differentially expressed in endothelial cells and in highly vascularised tissues [47], but no distinct role for PTGFR-v1 or PTGFR-v2 has been identified in relation to an altered cellular environment associated with pregnancy [48]. Ultimately, the functional role of each pair of wild-type GPCR and its truncated splice variant has to be studied under both normal and pathophysiological conditions, but proving a specific role for these highly truncated receptors will remain problematic when there is insufficient sequence difference between splice variant and wild-type GPCR mRNA to selectively knock down the splice variant using siRNA techniques.
Association of truncated GPCRs with constitutively active counterparts
The ghrelin receptor (GHS-R1a) and histamine H3 and H4 receptors are constitutively active and therefore can function independent of agonist [39, 40, 97], and dominant-negative effects have been observed with their truncated splice variants (see Table 1). The human H4 splice variant is differentially expressed in CD34+ cord blood-derived eosinophils and mast cells, but the functional consequence of heterodimerization with wild-type H4 is presently unknown [40]. For the ghrelin receptor, we see marked effects of its truncated 5TM splice variant (GHS-R1b) on constitutive activation of phospholipase C, with no effect on activation of ERK1/2 [36, 58, 98]. Bioluminescence resonance energy transfer (BRET) studies suggested that heterodimers of GHS-R1a/GHS-R1b are concentrated in the ER, whereas homodimers of GHS-R1a are more uniformly distributed throughout the cell [58]. It has been proposed that conflicting findings concerning the role of ghrelin in different tissues could be linked to the presence of this ghrelin receptor polypeptide GHS-R1b [14, 99]. There is no correlation between the expression of GHS-R1a mRNA and GHS-R1b mRNA in different tissues [95], and the factors regulating the expression of ghrelin receptor isoforms are unknown. The constitutive activity of the ghrelin receptor attenuates apoptosis via a protein kinase C-dependent process in vitro[100], and because GHS-R1b has a dominant-negative effect on ghrelin receptor function, any changes which increase GHS-R1b relative to GHS-R1a would be predicted to have a functional effect.
For an individual receptor, it is unclear to what degree such ligand-independent receptor signaling is present in the in vivo situation and consequently whether the constitutive signaling is of physiological relevance [101]. Some clues can be gained from other natural mutations of the ghrelin receptor and the melanocortin (MC4) receptor. Two families have been identified in which short stature is segregated with a GHSR mutation (Ala204Glu) that is characterized by substantially decreased basal activity of the receptor [102], possibly due to decreased expression in vivo[103]. Of the individuals who were identified as heterozygous for this mutation, not all had short stature. This observation is compatible with codominant transmission of the trait, with incomplete penetrance of the phenotype [102]. The constitutive activity of the MC4 receptor is maintained by its N-terminal domain, and mutations in the N-terminal lead to loss of basal activity and functional defects [104]. Obesity-associated mutations in the N-terminal domain of MC4 decrease its constitutive activity, which suggests that in addition to the agonist-mediated activation of MC4, this constitutive activity is also required for the maintenance of the anorexigenic catabolic state and the prevention of obesity in humans [104]. Together, these observations suggest that the physiological mechanisms controlled by ghrelin receptors and MC4 would be highly sensitive to the level of their basal activity. Since co-expression of ghrelin receptors and GHS-R1b in HEK293 cells dramatically decreases constitutive activation of phospholipase C [36, 98], and that this effect may be physiologically relevant, we have proposed that alternative splicing of genes for other constitutively active GPCRs will also dramatically affect cell signaling and functional activity.
Generation of receptor signaling complexes with altered pharmacology
Despite this dominant property of truncated GPCRs acting as dominant-negative mutants, there are examples where some functional activity is maintained. A 6TM μ-opioid receptor (MOR-1) variant in mice can identify ligands which lack the traditional side effects of classical opiates but maintain significant analgesic properties [6], and a 5TM somatostatin (sst2) receptor also retains functional activity and generates a receptor displaying an altered pharmacology [49]. A large series of splice variants of μ-OR (MOR) have been isolated from mice, rats, and humans with similar splicing patterns [6]. Most MOR-1 knock-out mice with a disruption of exon 1 were unresponsive to morphine, but mice with a series of MOR-1 variants generated from a second, upstream promoter associated with exon 11 had quite different pharmacology. These exon 11-associated variants lacking exon 1 are 6TM truncated variants, lacking the first TM domain of MOR-1 encoded by exon 1. One proposed partner for this inactive 6TM MOR-1 variant is the nociceptin (NOP) receptor [6]. By partnering of the mutant MOR-1 with NOP, the resulting heterodimers have a unique pharmacology which may provide valuable therapeutic targets.
The ghrelin receptor polypeptide (GHS-R1b) is another example of a functionally inactive truncated GPCR which can generate novel pharmacology by heterodimerization with a related GPCR. In this example, GHS-R1b can heterodimerize with the neurotensin receptor 1 (NTS1) to provide a receptor capable of responding to neuromedin U [7]. Treatment of non-small cell lung cancer cells with siRNA for GHS-R1b or NTS1 suppressed their growth in response to autocrine production of neuromedin U [7]. Indeed, when the ghrelin receptor splice variant GHS-R1b was first discovered [14], there was a rash of papers profiling GHS-R1a and GHS-R1b mRNA expression in human tumours, attempting to link altered expression with the state of malignancy [105–110]. It might be fruitful instead to look for potential novel partners for truncated splice variants in cells and tissues where conventional molecular tools have failed to compliment pharmacological identification of receptor subtypes.
By analogy with GPCR heterodimerization as a means to expand the repertoire of cell signaling [111], we can see that the availability of splice variant protomers will similarly influence the cell signaling capacity of GPCRs. It is well established that the majority of GPCRs are desensitized, internalized and down-regulated by constant or repeated exposure to agonist. And, it is now established that trafficking of GPCRs to the cell surface is a highly regulated process, and heavily influenced by the expression of highly truncated splice variants. Is it possible that expression of these splice variants allows fine-tuning of these processes? Is it possible that activation of a GPCR 'auto regulates' expression of its spliced isoforms or regulates the expression of other GPCR isoforms? At the present time, it is premature to answer these intriguing questions. The expectation of dominant-negative mutants to inactivate cell signaling by retaining the functional wild-type GPCR in the ER is perhaps an oversimplification because retention in the ER does not necessarily mean a loss of cell signaling activity. Furthermore, splice variants with 5TM or 6TM domains can be functionally active and present unique pharmacology by forming heterodimers with 7TM domain receptors. We could start by looking for potential novel partners for truncated splice variants in cells and tissues where conventional molecular tools have failed to compliment pharmacological identification of receptor subtypes. Currently, the incidence and association of splice variants with disease is an area of intense interest as a means to better target GPCR-dependent therapies. However, the association between GPCR polymorphisms and clinical disease are currently too weak to allow clinically meaningful predictions of GPCR variants and their relationship to disease onset or progression, or in drug responses [112]. Perhaps now we need to concentrate on treating each GPCR as a unique entity and thoroughly assess its activity profile in relation to the co-expression of splice variants in a cell-dependent and a time-dependent manner.
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- Holst B, Cygankiewicz A, Jensen TH, Ankersen M, Schwartz TW: High constitutive signaling of the ghrelin receptor - identification of a potent inverse agonist. Mol Endocrinol 2003, 17:2201–2210.PubMedGoogle Scholar
- Chu KM, Chow KBS, Leung PK, Lau PN, Chan CB, Cheng CHK, et al.: Over-expression of the truncated ghrelin receptor polypeptide attenuates the constitutive activation of phosphatidylinositol-specific phospholipase C by ghrelin receptors but has no effect on ghrelin-stimulated extracellular signal-regulated kinase 1/2 activity. Int J Biochem Cell Biol 2007, 39:752–764.PubMedGoogle Scholar
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Fashion-forward dress label Little Mistress offers the ideal style solution for the perfect bridesmaid dress or wedding guest outfit.
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Morning, Here's The First Trailer For The New Ghostbusters Movie It's here!
Valve Finally Notifies Steam Users About The Christmas Breach What a rapid response!
No Man's Sky Is Actually Coming Out Soon Yes. Please.
Bloodborne Cosplay Is Coming To Kick Your Arse Also yes please.
Spider-Man Is Totally OK With Letting Someone Else Be Spider-Man My son will be so relieved.


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"The Fiscal Year 2015 budget proposal released today by the Senate Committee on Ways and Means invests in important priorities such as special education, substance abuse, child welfare, and public safety. In addition the committee has made laudable strides in the proposal to reduce its dependence on the stabilization fund and increasing taxes.
Today's action by the committee is an important starting point, and in the days ahead we must work vigorously to secure initiatives to grow jobs, reform state government, and find savings and efficiencies to complete the effort.
Our caucus will be offering many ways to do so, and we look forward to working with our colleagues to build a post-recession Massachusetts that has efficiency and fiscal discipline in state government, and an economic climate that promotes prosperity for years to come."


text: Property uninhabitable due to fire. 2-family zoned LOT 'by right' in heart of Melrose! This 11,120 sq. ft. lot offers unique development opportunity in one of the hottest zip codes in the country! COMPARABLE SALE: A lot less than half the size in a less desirable location with a shared driveway and compromised soil recently sold in the mid $500s. Yielding a walk score of 87, this location offers easy access to public transportation to commuter rail and Oak Grove T. The heart of Downtown Main Street Melrose is a block away...Turner's Seafood, Starbucks, CVS, Brueggers, boutique shops and more!


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Mobile Marketing in India takes a U-turn
Remember the likes of sms based advertising models where you register free to receive SMS's from 'advertisers' and earn money, etc; namely, m gingur, adcadmobi, etc... There seems to be another player in the market who is trying to make this loss-stricken idea into a beneficial one. The company '160by2' claims to give you the ability to register for free, earn money for receiving SMS's from their advertisers but the new marketing strategy that they have adopted is that users (like you and me) can use 80 characters to Send Free Messages to all mobile numbers India and the next 80 characters will contain an advert from one of the advertisers of 160by2 company.
Unfortunately, what the promoters have seemed to ignore is the reason why earlier business models like these failed - Ding Dong! MGingur and the likes, did mass marketing and advertising with a lot of ( I repeat a lot of ) word of mouth marketing which ended up approx 5 lac users being registered. The limitation of this model is that most advertisers (in India and the world) seek information of people or to target people who have reasonable disposable income and are willing to spend it. But the data collected in these campaigns or the people targetted by SMS marketing are of the nature who want to make money by collecting a few paise (smallest unit of the INR) would not be the kinds of to either have reasonable disposable income nor be willing to spend much. Nevertheless, this segment of people are quite good for targetting goods like Coke, Chocolates or items of not much monetary value. On the contrary, not many (or maybe not at all) of these type of companies would be interested in promoting their products by Direct marketing methods like SMS Marketing. Wake up and smell the coffee guys.


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Hello everyone (again), im going to be sharing with you the nails im wearing today and will be wearing for the next couple days.
|My left hand...
|and my right hand.
So all I done was is paint my middle finger, ring finer and thumb one colour and, my index and pinky another. Also just to add abit more of a girly touch to it I put a glitter opolish over the top on my ring finger on each hand.


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Thiomucase is a gel that claims to reduce the appearance of cellulite, and it recently underwent a rebranding that was led by the creative agency Metaklinika.
The purpose of rebranding this product was to give it a "clean and fresh" appearance, one that would ensure that it still offers a "medical" aesthetic, while also offering a contemporary feel. The creative agency chose to make the product look less like a pharmaceutical item and more like a premium cosmetic product, while still offering clean lines, limited labeling and sparse coloring.
The gel itself is extremely popular in Southern Europe, and is applied topically on areas that tend to have cellulite – working to boost circulation and reduce the appearance of that cellulite as a result.
Premium Anti-Cellulite Gels
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App-Controlled Smart Bandages
Alcoholic Gel Capsules
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Thiomucase Has Redesigned Its Branding
- By: Mishal OmarOct 13, 2017
References: metaklinika & thedieline
References: metaklinika & thedieline


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BaByliss Pro Clipper Steel FX Supercharged Pivot Motor #FX880
The powerful BaBylissPRO FX880 professional clipper features a high-frequency pivot motor, stainless steel blades, 5-position detent taper control, 45 length settings and 8 comb attachments. It's scientifically balanced for superior ergonomics, and the all-metal housing is designed for uncompromising integrity. Perfect for cutting all hair types; can be used wet or dry.


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Countryside wardens at Guisborough Forest and Walkway will lead a volunteer taster day to go 'bramble bashing'.
Those wanting to take part in the conservation task, which involves clearing bramble bushes along a section of the old railway line, are advised to wear old clothes and take a packed lunch, a hot drink and waterproofs. Stout footwear is also recommended.
The event, on Saturday January 24 from 10am to 3pm, is part of a programme by Redcar and Cleveland Council's dedicated band of countryside volunteers. All tools and equipment will be provided.
Young people, aged 12 to 16, will need a consent form. Call 01287 631132 for more details.


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Found 3 words containing bandoneo. Browse our Scrabble Word Finder, Words With Friends cheat dictionary, and WordHub word solver to find words that contain bandoneo. Or use our Unscramble word solver to find your best possible play! Related: Words that start with bandoneo
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In a small river village where the water is cursed, a girl's bravery could mean the difference between life and death in this lilting, luminous tale from the author of Parched and Audacity.
Along a lively river, in a village raised on stilts, lives a girl named Luna. All her life she has heard tales of the time before the dam appeared, when sprites danced in the currents and no one got the mysterious wasting illness from a mouthful of river water. These are just stories, though—no sensible person would believe in such things.
Beneath the waves is someone who might disagree. Perdita is a young water sprite, delighting in the wet splash and sparkle, and sad about the day her people will finally finish building their door to another world, in search of a place that humans have not yet discovered.
But when Luna's little sister falls ill with the river sickness, everyone knows she has only three weeks to live. Luna is determined to find a cure for her beloved sister, no matter what it takes. Even if that means believing in magic…
REVIEW BY: Michaela, age 11 years, 3 months
This book was sad, but very good. There were a couple parts that made me cry.
This story has three different sides to it, those being the spirit twins and Luna.
My favorite character is Willow because she is strong even when she is sick. My favorite part is when Luna meets Gia, who the swamp people say makes them sick.
This book is full of magic in the swamp. What more could you ask for in a book?
I give this book 5 out of 5 stars and recommend it for ages 10-13 years old.
This book was published on September 8, 2015.


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Celebrate memories that last a lifetime with our family necklace inspired by simple moments spent together by the coast. Each beach necklace is delicately etched with a family of four walking hand in hand along the golden sand.
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Getting experienced from anorexia for a few years After i was young, I am combating this type of large gain. What must I do?
wikiHow Contributor It is dependent Everything you necessarily mean by "rapidly". A constant rapidly isn't a good suggestion, as you may deny the infant several of the nutrients it needs for advancement. Conversely, half-working day fasts for a short time frame is likely to be fine, supplied you consume very well and consume an abundance of drinking water throughout the non-fasting Portion of the day and acquire a good amount of rest and stay away from arduous action.
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|Religious Reformation
|Written by Kris Brewer
|Friday, 22 October 2010 19:46
Recently, we have been studying the Minor Prophets in our Adult Bible study on Sunday mornings. We recently started a study of the prophet Micah. As I was preparing to teach this lesson, I came upon a quote by Homer Hailey in his commentary on the Minor Prophets. He was commenting on the nature of the people during the time of Micah, and how they were not what they should have been, even though they had some pretty good kings (such as Jotham and Hezekiah). Hailey's analysis on the matter was this:
"Micah seems not to have been very deeply impressed with these reforms. Religion had become a matter of form with the people; ceremonial observances were thought to meet all religious requirements. The reform was not from the deep recesses of the heart. There was widespread misapprehension that as long as the external acts of worship were scrupulously performed the people were entitled to the divine favor and protection."
Hailey's observation is one that could probably be made for many people today! Religion has certainly become a matter of form for many people, with some type of ceremonial observance being more important than being truly obedient to God. To a greater extent (at least in the denominational world) we can see those ceremonial observances being things that are completely foreign to the word of God. There have been additions made to the instructions left to us by God, which many believe must be accepted just because they are offered to Him. In reality, we need to understand that these "ceremonies" are not going to save our souls and they are not going to force God to accept our worship or service.
To hit a bit closer to home, I believe that many in the Lord's church have expressed the same kind of idea as the children of Israel in Micah's day. They have determined that the ceremonial action of taking the Lord's Supper is essentially the sole determinant of their faithfulness before God! I have seen professed Christians rush in to a church building at the moment that they knew the Lord's Supper would be offered, partake of it, and then rush out (completely ignoring the rest of the assembly, and their responsibilities to worship in other ways). They had determined that, on the whole, the things they had planned were of more importance than worshiping the the Lord, but they felt obligated to partake of the Lord's Supper! They obviously believed that the fulfillment of that "ceremonial observance" allowed them to meet all of their "religious requirements."
While partaking of the Lord's Supper is indeed a very important responsibility of the modern church, we should never get to the point that we think of such practices as some kind of a ceremonial marker to faithfulness. Faithful Christians will partake of the Lord's Supper, but partaking of the Lord's Supper is not what makes one a faithful Christian! There are buildings full of people who will take the Lord's Supper, but are not faithful to God's word! We need to make sure that we are not only doing what we ought (fulfilling the practices that are required) but that we are doing them for the right reasons! If we offer our sacrifice for the wrong reasons, then we are no better off than the children of Israel who offered their burnt offerings, but then turned around and served the false gods as well.
God is no way required, or obligated to accept our worship. Only when we present to Him what He has asked for will he accept it. We must not succumb to the idea that we can present to God whatever makes us feel good, whatever we desire for our own selfish ambitions, and He will accept it without question. He has revealed His mind for one reason: So that we will know what to do, and how to do it. This is the only way that we can be pleasing to Him!
So, if we are going to be "religious reformers," directing men today back to the practices and teachings of the New Testament church, we must dedicate ourselves to true reform. We must make changes in the "deep recesses of the heart," committing ourselves to God on every level. Just going through the motions is not going to cut it! It is our obligation to seek out the will of God, and put all that He requires into practice. Do not fool yourself into believing that there is a simple checklist that identifies you as being faithful. There is an obligation to live a life for God, not just perform a few repetitive tasks! See Romans 12:1-2.
|Last Updated on Friday, 22 October 2010 19:52
Are you looking for the church of Christ in Huntington? You can visit that website by clicking here.
Looking for the Gallatin Valley church of Christ? You can visit that website by clicking here.
This is the personal page of Kris Brewer, who currently works with the Gallatin Valley church of Christ in Bozeman, Montana.


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The Digital Elevation Model of Indiana - Revised CD-ROM contains a 30-meter DEM based on the U.S. Geological Survey's National Elevation Dataset (NED) for Indiana and the surrounding states. IGS reconstruction has modified much of the original data set. An explanation of the process used to make new DEMs and images of the final grid are included on the CD-ROM.
Brown, S. E., Bleuer, N. K., Dickson, M. L., Dintaman, C. , Olejnik, J. , Rupp, R. F. 2004, Digital Elevation Model of Indiana—Revised: Indiana Geological Survey Open-File Study 04-01
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Keywords: GIS, digital elevation model, DEM, digital mapping, landforms, land surface, terrain elevation, topography
Can't find what you're looking for? Feel free to contact us directly:
Publication Sales Office
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Bloomington, IN 47405-2208
812-855-7636 (phone)
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IGS Return Policy
All sales are final. Materials may not be returned after they have been accepted by the customer. Any discrepencies in mail orders must be reported to IGWS within 10 days of receipt of order.
The Indiana Geological and Water Survey will refund the purchase price and sales tax of products if returned with the original sales receipt within 10 days of receipt of order. Refunds will be issued with the same method of payment as indicated on the original receipt except for cash purchases, which will be refunded by check. Items must be returned in the same condition as purchased. Refunds on digital products and services are not allowed, except for defective media such as CD-ROMs, flash drives, and so on. The customer is responsible for paying shipping costs to return items.


text: So I just recently received my brand new 10GB iPod and I have a question about the playlists. So you can set it to update the all songs and playlists....but i have too many songs for that. You can set it to update selected playlists.....or you can set it to manually manage songs. So what I put the playlists I wanted on my iPod and then began adding songs one at a time. Now I've created another playlist and want to add that to my iPod. How do I add just the one playlist to the iPod without removing all of the songs I put on my iPod one at a time? Thanks.


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Tom Coronel fined for unsporting behavior
ROAL Motorsport's driver Tom Coronel has been fined by the FIA for unsporting behavior during WTCC weekend at Vila Real in Portugal.
The Dutch driver insulted Ma Qing Hua after the qualifying session and accused the Citroen driver for blocking him. Stewards investigated the situation and concluded that Ma Qing Hua made peace was as soon as he could. Coronel later apologised, but it was too late as officials decided that abusive language is not acceptable in an FIA Championship.
That's why Coronel has been fined with 1000 euros and was ordered to perform some public acitivity for the FIA. ROAL Motorsport's driver wasn't happy with outcome: "They said I should not call him a stupid driver and things like this; they said they don't want that. So I have a fine and for the FIA I have to do a schooling thing or an event. So, it seems I'm not allowed to say to someone if he's stupid."
Citroen Racing team principal Yves Matton commented that a fine for Tom Coronel is fair and completely deserved as his behavior was completely unacceptable.


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Job Description
From what I gather, if there were ever a job description typed out for a farmer, I think it would look something like this:
- To provide food for 155 people annually
- Provide food, shelter, water, health care, and attention to their livestock every day
- Be environmental stewards
- Ensure that America's food is healthy, great-tasting, and safe, free of drugs or harmful chemicals or disease
Qualifications and Skills:
- Physical capabilities of lifting upwards of 100 lbs
- Mechanical skills to fix farm equipment without a shop
- Accounting skills to manage the operation's funds
- Carpentry, Plumbing and Construction skills to build fences, repair water lines, etc to maintain animal facilities
- Record keeping, to great detail, to provide food traceability
- Basic veterinary skills to help birthing, give accurate dosages of vaccines and medications, minor surgical skills, etc
- Stay updated on laws passed in the areas of animal welfare, soil, waste management, fertilizers, vaccines, medications, withdrawl times, etc
Work Conditions
- Your hours will be 7 days a week, an average of 10-12 hours a day
- Sundays you may be able to get by with 3-4 hours, just enough to feed and check up on the animals
- During calving/lambing/farrowing season, you may be sleeping in the barn or waking up every 2 hours to check on the mothers and babies
- No paid holidays or sick days. In fact, if you miss any days, you will lose pay. Even if you are sore, sick, or puking, you must take a few hours to at least feed the animals. Unless you can find someone else to do it for you. If you do not have family members who can fill in, you may be out of luck.
- If you would still like a day off, you are responsibly for finding your substitute and paying them if necessary. (If you think finding a pet-sitter for your 2 dogs is hard, imagine trying to find someone to go outside in -10°F weather and feed 500 animals)
- You will likely work outside most hours of your shifts. The temperatures will range from 110°F to -20ºF.
- You may come under fire from media who do not understand your lifestyle and practices.
- Unlike those who provide safety to our country (such as police, military, firemen), you may never get thanked for providing one of the biggest human necessities of life: Food.
- There are no health benefits, 401k matching programs, or company discounts. (unless you count the meat you keep instead of selling for profit)
- Your average net income will be less than $20,000/year, so please prepare to work a second job. This income is contingent upon the weather, prices of gasoline, corn, and other feedstuffs. Please note that if you manage to do all your work in just 60 hours a week, this averages to around $6.00/hour.
So, anyone still interested?
At the very least, I bet you're no longer surprised that less than 2% of America loves their land and animals enough to sign up for this kind of work environment. That really says something about that 2% though, doesn't it?
Posted on February 18, 2011, in Ag-the-Culture and tagged Farmers, Work Conditions. Bookmark the permalink. 10 Comments.
This is a great blog that you have going here, you now have my e-mail and as an Agriculturist I will be glad to help you with anything that you may need. This job description is actually quite scary, I mean why in the world did i sign up for this job (oh, it probably had quite a bit to do with the love and compassion for animals that all of us agriculturists have). I do have one correction for you though. The American Farm Bureau states that the average farmer produced enough food in 2010 to feed 155 people. Here is the link http://www.fb.org/index.php?fuseaction=yourag.facts
Interesting post topic. Enlightening, too.
Really cool post definitely going to post it on my facebook page later today. You can search crystal cattle.
Awesome, awesome post. You portrayed perfectly what it means to feed the world. Thank you, farmers!
True,add about 20hrs to your week…lol
This is awesome. This really needs to go out to every internet site possible. Every politician should also have a copy.
Thank you so much for this.


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Naval Ship Research and Development Center, Bethesda, Md.
A nonlinear method of finding two-dimensional supercavitating sections in a cascade is developed. The load distribution is taken from linear theory for low drag super-cavitating foils in a cascade. The double spiral vortex model is used for the cavity termination. Linear and nonlinear theories are compared with special emphasis put on analyzing the blunt leading edge. The foil-cavity shape, the cavity drag and the cavitation number can be obtained by a computer program from the given cavity length and geometrical parameters of the cascade.
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This Is It - My Final Curtain
A collection of original short stories, poetry, and interviews
Bargin Books, Games, Music, Movies & More
Thursday, May 17, 2012
A Slave
. . . A slave is bound to remind her of the ties that entwine them.
. . . She is placed in the dark so she may see the light when her Master speaks.
. . . She is adored.
. . . She is treasured.
. . . She is owned.
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We had the opportunity to share a couple of creative arts, worship nights with our community groups over the past couple of weeks. They have been a wonderful opportunity to share the unique gifts that God has given us with one another. We have had the opportunity to hear God's truth communicated through folk music, and rap, spoken word and 19th century poetry, as well as painting and Chinese alphabet characters. Here are a couple of pictures from our most recent event, the Unveiling.
I shared two spoken word poems at the recent event. A couple of people asked to get a copy of them, so I will attach the audio below. The first is a reflection on the way that even though I didn't know about Advent growing up, it was still a meaningful description of the way I experienced December as a child. The second is an invitation to return to the historic practice of celebrating Christmas as a 12 day season, rather than a one day event. I hope you enjoy them.
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Author: Cameron Lemons
Reflections from the pastor
March 2017