The CRMP (collapsin response mediator protein) family is thought to play key roles in growth cone guidance during neural development. The four members (CRMP1-4) identified to date have been demonstrated to form hetero-multimeric structures through mutual associations. In this study, we cloned a novel member of this family, which we call CRMP5, by the yeast twohybrid method. This protein shares relatively low amino acid identity with the other CRMP members (49 -50%) and also with dihydropyrimidinase (51%), whereas CRMP1-4 exhibit higher identity with each other (68 -75%), suggesting that CRMP5 might be categorized into a third subfamily. The mouse CRMP5 gene was located at chromosome 5 B1. Northern blot and in situ hybridization analyses indicated that CRMP5 is expressed throughout the nervous system similarly to the other members (especially CRMP1 and CRMP4) with the expression peak in the first postnatal week. Association experiments using the yeast two-hybrid method and coimmunoprecipitation showed that CRMP5 interacts with dihydropyrimidinase and all the CRMPs including itself, except for CRMP1, although the expression profile almost overlaps with that of CRMP1 during development. These results suggest that CRMP complexes in the developing nervous system are classifiable into two populations that contain either CRMP1 or CRMP5. This indicates that different complexes may have distinct functions in shaping the neural networks.

It is well documented that grain feeding stimulates adipogenesis in beef cattle, whereas pasture feeding depresses the development of adipose tissues, including intramuscular (i.m.) adipose tissue. Additionally, production practices that depress adipocyte differentiation also limit the synthesis of MUFA. Marbling scores and MUFA increase in parallel suggesting that stearoyl-coenzyme A desaturase (SCD) gene expression is closely associated with and necessary for marbling adipocyte differentiation. Similarly, marbling scores and fatty acid indices of SCD activity are depressed in response to dietary vitamin A restriction. In bovine preadipocytes, vitamins A and D both decrease glycerol-3-phosphate dehydrogenase (GPDH) activity, an index of adipocyte differentiation, whereas incubation of bovine preadipocytes with l-ascorbic acid-2-phosphate increases GPDH activity. Exposing bovine preadipocytes to zinc also stimulates adipogenesis, putatively by inhibiting nitric oxide (NO) production. However, incubation of bovine preadipocytes with arginine, a biological precursor of NO, strongly promotes differentiation in concert with increased SCD expression. This suggests that the effect of either arginine or zinc on adipogenesis is independent of NO synthesis in bovine preadipocytes. Enhanced expression of SCD is associated with a greater accumulation of MUFA both in bovine preadipocyte cultures and during development in growing steers. In bovine preadipocytes, trans-10, cis-12 CLA strongly depresses adipocyte differentiation and SCD gene expression, thereby reducing MUFA concentrations. The bovine preadipocyte culture studies suggest that any production practice that elevates vitamins A or D or trans-10, cis-12 CLA in bovine adipose tissue will reduce i.m. adipose tissue development. Conversely, supplementation with vitamin C or zinc may promote the development of i.m. adipose tissue.

Angus and Wagyu steers consuming high-roughage diets exhibit large differences in adipose tissue fatty acid composition, but there are no differences in terminal measures of stearoyl-CoA desaturase (SCD) activity or gene expression. Also, adipose tissue lipids of cattle fed corn-based diets have greater MUFA:SFA ratios than cattle fed hay-based diets. We hypothesized that any changes in SCD gene expression and activity would precede similar changes in adipose tissue lipogenesis between short- and long-fed endpoints. Furthermore, changes in SCD activity and gene expression between production endpoints would differ between corn- and hay-fed steers and between Wagyu and Angus steers. Angus (n = 8) and Wagyu (n = 8) steers were fed a corn-based diet for 8 mo (short-fed; 16 mo of age) or 16 mo (long-fed; 24 mo of age), whereas another group of Angus (n = 8) and Wagyu (n = 8) steers was fed a hay-based diet for 12 mo (short-fed; 20 mo of age) or 20 mo (long-fed; 28 mo of age) to match the end point BW of the corn-fed steers. Acetate incorporation into lipids in vitro was greater (P < 0.01) in corn-fed steers than in hay-fed steers and tended (P = 0.06) to be greater in Wagyu than in Angus s.c. adipose tissue because the rate in Wagyu was twice that of Angus adipose tissue in the corn-fed, short-fed steers. There were diet x end point interactions for lipogenesis in i.m. and s.c. adipose tissues (both P < 0.01) because lipogenesis was 60 to 90% lower in the long-fed cattle than in short-fed cattle fed the corn-based diet. The greatest SCD enzyme activity in Angus s.c. adipose tissue was observed at 24 mo of age (corn-based diet), but activity in Wagyu adipose tissue was greatest at 28 mo of age (hay-based diet; breed x diet x end point interaction, P = 0.08). For short- vs. long-fed endpoints in Angus, s.c. adipose tissue SCD activity was less (hay diet) or the same (corn diet). Conversely, SCD gene expression was greatest in long-fed Wagyu steers fed the hay- or corn-based diets (breed x end point interaction; P < 0.01). Contrary to our hypotheses, SCD activity increased over time, whereas lipogenesis from acetate decreased. However, the developmental pattern of SCD gene expression and activity differed markedly between hay-fed Angus and Wagyu adipose tissues, which may explain the differences in the MUFA:SFA ratios observed in adipose tissues from these cattle.

We used a genetic method, the yeast substrate-trapping system, to identify substrates for protein tyrosine phosphatases (PTP͞ RPTP␤). This method is based on the yeast two-hybrid system, with two essential modifications: conditional expression of protein tyrosine kinase v-src (active src) to tyrosine-phosphorylate the prey proteins and screening by using a substrate-trap mutant of PTP (PTP-D1902A) as bait. By using this system, several substrate candidates for PTP were isolated. Among them, GIT1͞Cat-1 (G protein-coupled receptor kinase-interactor 1͞Cool-associated, tyrosine-phosphorylated 1) was examined further. GIT1͞Cat-1 bound to PTP-D1902A dependent on the substrate tyrosine phosphorylation. Tyrosine-phosphorylated GIT1͞Cat-1 was dephosphorylated by PTP in vitro. Immunoprecipitation experiments indicated that PTP-D1902A and GIT1͞Cat-1 form a stable complex also in mammalian cells. Immunohistochemical analyses revealed that PTP and GIT1͞Cat-1 were colocalized in the processes of pyramidal cells in the hippocampus and neocortex in rat brain. Subcellular colocalization was further verified in the growth cones of mossy fibers from pontine explants and in the ruffling membranes and processes of B103 neuroblastoma cells. Moreover, pleiotrophin, a ligand for PTP, increased tyrosine phosphorylation of GIT1͞Cat-1 in B103 cells. All these results indicate that GIT1͞Cat-1 is a substrate molecule of PTP.

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In current models of transforming growth factor-β (TGF-β) family signaling, type II receptors activate specific activin receptor-like kinase (ALK) type I receptors. These serine/threonine kinases activate ligand-dependent receptor regulated (R)-Smad by phosphorylating carboxyterminal serines. We found that the receptor expression levels affected the phosphorylation and activation of the two R-Smad subclasses, activin/TGF-b-specific (AR-Smad) and bone morphogenetic protein (BMP)-specific (BR-Smad). Co-expressing constitutively active type I and type II receptors in COS7 cells resulted in the phosphorylation of both R-Smad subclasses in a ligand-independent manner. This was verified using in vitro kinase assays. In untransfected B16 melanoma cells, TGF-β1 and BMP-2 induced phosphorylation of both R-Smad subclasses, and TGF-b1 up-regulated the inhibitor of differentiation (Id) gene, which is usually regulated by BMP. By contrast, BMP-2 up-regulated plasminogen activator inhibitor-1 (PAI-1), which is an AR-Smad-regulated gene. Except for ALK4 and ALK6, levels of type I and type II receptor mRNAs were higher in B16 cells than in HeLa and HepG2 cells, in which TGF-b1 and BMP-2 induced phosphorylation of only the expected R-Smad. These results help to explain the diverse effects of this ligand family.

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