Protection of transforming growth factor β activity by heparin and fucoidan

The transforming growth factor-β (TGF-β) family of proteins exert diverse and potent effects on proliferation, differentiation, and extracellular matrix synthesis. However, relatively little is known about the stability or processing of endogenous TGF-β activity in vitro or in vivo. Our previous work indicated that (1) TGF-β1 has strong heparin-binding properties that were not previously recognized because of neutralization by iodination, and (2) heparin, and certain other polyanions, could block the binding of TGF-β1 to α₂-macroglobulin (α₂-M). The present studies investigated the influence of heparin-like molecules on the stability of the TGF-β1 signal in the pericellular environment. The results indicate that heparin and fucoidan, a naturally occurring sulfated L-fucose polymer, suppress the formation of an initial non-covalent interaction between ₁₂₅I-TGF-β1 and activated α₂-M. Electrophoresis of ₁₂₅I-TGF-β1 showed that fucoidan protects TGF-β1 from proteolytic degradation by plasmin and trypsin. While plasmin caused little, if any, activation of latent TGF-β derived from vascular smooth muscle cells (SMC), plasmin degraded acid-activated TGF-β, and purified TGF-β1, and this degradation was inhibited by fucoidan. In vitro, heparin and fucoidan tripled the half-life of ₁₂₅I-TGF-β1 and doubled the amount of cell-associated ₁₂₅I-TGF-β1. Consistent with this protective effect, heparin- and fucoidan-treated SMC demonstrated elevated levels of active, but not latent, TGF-β activity. © 1994 wiley-Liss, Inc.     

Visualization and ablation of phenylethanolamineN-methyltransferase producing cells in transgenic mice

We cloned and sequenced the mouse phenylethanolamineN-methyltransferase (PNMT) gene which encodes the enzyme that catalyses the conversion of norepinephrine to epinephrine. The ability of various length sequences flanking the mouse or human PNMT genes to direct expression of reporter genes in transgenic mice was examined. We show that 9 kb of 5 flanking sequences from the cloned mouse PNMT gene can direct expression of theEscherichia coli -galactosidase (lacZ) gene to predicted regions of the adrenal, eye can direct in the adult transgenic mouse. The transgene was also expressed during development, in the myelencephalon, adrenal medulla and dorsal root ganglia. PNMT-producing cells were ablated by expression of the diphtheria toxin (DT-A) gene driven by the human PNMT promoter, resulting in abnormalities in the adrenal medulla, eye and testis. The hPNMT_{8 kb}-DT-A line presents a model with which to examine the developmental ramifications of deletion of PNMT-producing cell populations from the adrenal medulla and retina.     

Cell cycle regulation of the c-Myc transcriptional activation domain.

The product of the c-myc gene (c-Myc) is a sequence-specific DNA-binding protein that has previously been demonstrated to be required for cell cycle progression. Here we report that the c-Myc DNA binding site confers cell cycle regulation to a reporter gene in Chinese hamster ovary cells. The observed transactivation was biphasic with a small increase in G1 and a marked increase during the S-to-G2/M transition of the cell cycle. This cell cycle regulation of transactivation potential is accounted for, in part, by regulatory phosphorylation of the c-Myc transactivation domain. Together, these data demonstrate that c-Myc may have an important role in the progression of cells through both the G1 and G2 phases of the cell cycle.     

The detection and attribution of extreme reductions in vegetation growth across the global land surface

Negative extreme anomalies in vegetation growth (NEGs) usually indicate severely impaired ecosystem services. These NEGs can result from diverse natural and anthropogenic causes, especially climate extremes (CEs). However, the relationship between NEGs and many types of CEs remains largely unknown at regional and global scales. Here, with satellite-derived vegetation index data and supporting tree-ring chronologies, we identify periods of NEGs from 1981 to 2015 across the global land surface. We find 70% of these NEGs are attributable to five types of CEs and their combinations, with compound CEs generally more detrimental than individual ones. More importantly, we find that dominant CEs for NEGs vary by biome and region. Specifically, cold and/or wet extremes dominate NEGs in temperate mountains and high latitudes, whereas soil drought and related compound extremes are primarily responsible for NEGs in wet tropical, arid and semi-arid regions. Key characteristics (e.g., the frequency, intensity and duration of CEs, and the vulnerability of vegetation) that determine the dominance of CEs are also region- and biome-dependent. For example, in the wet tropics, dominant individual CEs have both higher intensity and longer duration than non-dominant ones. However, in the dry tropics and some temperate regions, a longer CE duration is more important than higher intensity. Our work provides the first global accounting of the attribution of NEGs to diverse climatic extremes. Our analysis has important implications for developing climate-specific disaster prevention and mitigation plans among different regions of the globe in a changing climate.     

Leaf nutrients in relation to stature and life form in tropical rain forest

Abstract. To document the relationship between a plant's position in the canopy and its leaf nutrient content, leaf nitrogen and phosphorus were determined for 30 species growing in mature evergreen lowland rain forest at La Selva Biological Station, Costa Rica. Species that grow either in the understory, midstory, or the canopy were selected. Species were further separated into three life forms: self-supporting monocots, self-supporting dicots, and climbers. Mass-based nutrient concentrations were expected to decrease with stature, as has been reported in studies of other forests. In fact, mass-based nitrogen and phosphorus did not vary significantly among the three adult-stature classes, although area-based values differed greatly: canopy plants averaged 60 % more nitrogen and 90 % more phosphorus per unit leaf area than understory plants. Differences in leaf characteristics were evident among the three life forms. Most notably, area-based phosphorus and leaf specific mass were lowest in climbers, intermediate in self-supporting dicots, and highest in self-supporting monocots. These results support the characterization of climbers as investing in inexpensive structures, perhaps in order to gain competitive advantage in light capture by allocating resources to maximize elongation rates.     

Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein kinase.

A growth factor-stimulated (MAP2-related) protein kinase, ERT, that phosphorylates the epidermal growth factor receptor at Thr669 has been purified from KB human tumor cells by Northwood and co-workers (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). The ERT protein kinase has a restricted substrate specificity, and the structural determinants employed for substrate recognition by this enzyme have not been defined. As an approach toward understanding the specificity of substrate phosphorylation, we have used an in vitro assay to identify additional substrates for the ERT protein kinase. In this report we describe two novel substrates: (a) the human c-myc protein at Ser62 and (b) the rat c-jun protein at Ser246. Alignment of the primary sequences surrounding the phosphorylation sites located within the epidermal growth factor receptor (Thr669), Myc (Ser62), and Jun (Ser246) demonstrated a marked similarity. The observed consensus sequence was Pro-Leu-Ser/Thr-Pro. We propose that this sequence forms part of a substrate structure that is recognized by the ERT protein kinase.     

Mutational analysis of the cytoplasmic tail of the human transferrin receptor. Identification of a sub-domain that is required for rapid endocytosis

It has been reported that the sequence Tyr20-X-Arg-Phe23 present within the cytoplasmic tail of the transferrin receptor may represent a tyrosine internalization signal (Collawn, J.F., Stangel, M., Kuhn, L.A., Esekogwu, V., Jing, S., Trowbridge, I.S., and Tainer, J. A. (1990) Cell 63, 1061-1072). However, as Tyr20 is not conserved between species (Alvarez, E., Gironès, N., and Davis, R. J. (1990) Biochem. J. 267, 31-35), the functional role of the putative tyrosine internalization signal is not clear. To address this question, we constructed a series of 32 deletions and point mutations within the cytoplasmic tail of the human transferrin receptor. The effect of these mutations on the apparent first order rate constant for receptor endocytosis was examined. It was found that the region of the cytoplasmic tail that is proximal to the transmembrane domain (residues 28-58) is dispensable for rapid endocytosis. In contrast, the distal region of the cytoplasmic tail (residues 1-27) was found to be both necessary and sufficient for the rapid internalization of the transferrin receptor. The region identified includes Tyr20-X-Arg-Phe23, but is significantly larger than this tetrapeptide. It is therefore likely that structural information in addition to the proposed tyrosine internalization signal is required for endocytosis. To test this hypothesis, we investigated whether a heterologous tyrosine internalization signal (from the low density lipoprotein receptor) could function to cause the rapid endocytosis of the transferrin receptor. It was observed that this heterologous tyrosine internalization signal did not allow rapid endocytosis. We conclude that the putative tyrosine internalization signal (Tyr20-Thr-Arg-Phe23) is not sufficient to determine rapid endocytosis of the transferrin receptor. The data reported here indicate that the transferrin receptor internalization signal is formed by a larger cytoplasmic tail structure located at the amino terminus of the receptor.