Grb2 mediates negative regulation of stem cell factor receptor/c-Kit signaling by recruitment of Cbl

Aberrant activation of c-Kit is involved in a number of human diseases including cancers and leukemias. Certain receptor tyrosine kinases, such as epidermal growth factor receptor, have been shown to indirectly recruit Cbl through the adapter protein Grb2, leading to receptor ubiquitination and degradation. In order to study the role of Grb2 in c-Kit degradation, a series of mutations of the Grb2 binding sites in c-Kit were generated (Y703F, Y936F, and Y703F/Y936F). Since other signal transduction molecules are also known to bind Y703 and Y936, the more selective asparagine-to-alanine (N-to-A) mutants N705A, N938A, and N705A/N938A were generated. We could clearly demonstrate that binding of Grb2 was dependent on intact phosphorylation sites Y703 and Y936. Furthermore, we could demonstrate the presence of Cbl in a complex with Grb2 and c-Kit. Thus, Grb2 is able to indirectly recruit Cbl to c-Kit. In the N-to-A mutants, Cbl phosphorylation was strongly reduced, which correlated with reduced ubiquitination of c-Kit as well as decreased internalization and degradation of the receptor. Taken together, we have demonstrated that, in addition to its role in positive signaling via the Ras/Erk pathway, Grb2 mediates c-Kit degradation through recruitment of Cbl to c-Kit, leading to ubiquitination of c-Kit followed by internalization and degradation.     

Somatostatin induces rapid contraction of neuroendocrine cells

The peptide hormone somatostatin, as well as the somatostatin analog octreotide, induces rapid morphological changes in neuroendocrine cells. The effect can be detected in less than 2 min: retraction fibers are formed, cells round up and cell-cell contacts are broken. Somatostatin-dependent cell contraction is inhibited by Y-27632, indicating that this effect is dependent on Rho kinase. In BON1 cells, the somatostatin-induced inhibition of forskolin-induced secretion of chromogranin A is not blocked by Y-27632. It is therefore concluded that the inhibitory effect of somatostatin in forskolin-stimulated cells is not dependent on cell contraction.     

Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity

Dissociated primary sensory neurons are commonly used to study growth factor-dependent cell survival, axon outgrowth, differentiation and basic mechanisms of sensory physiology and pain. Spinal or trigeminal sensory neurons can be collected from embryos, neonates or adults, treated with enzymes that degrade the extracellular matrix, triturated and grown in defined media with or without growth factors and additional animal sera. Production of cultures can take as little as 2.5 h. Cells can be used almost immediately or maintained for as long as 1 month. Ease of production and the ability to control growth conditions make sensory neuron culture a powerful model system for studying basic neurobiology of central and peripheral nervous systems.     

Phyllachora species infecting maize and other grass species in the Americas represents a complex of closely related species

The genus Phyllachora contains numerous obligate fungal parasites that produce raised, melanized structures called stromata on their plant hosts referred to as tar spot. Members of this genus are known to infect many grass species but generally do not cause significant damage or defoliation, with the exception of P. maydis which has emerged as an important pathogen of maize throughout the Americas, but the origin of this pathogen remains unknown. To date, species designations for Phyllachora have been based on host associations and morphology, and most species are assumed to be host specific. We assessed the sequence diversity of 186 single stroma isolates collected from 16 hosts representing 15 countries. Samples included both herbarium and contemporary strains that covered a temporal range from 1905 to 2019. These 186 isolates were grouped into five distinct species with strong bootstrap support. We found three closely related, but genetically distinct groups of Phyllachora are capable of infecting maize in the United States, we refer to these as the P. maydis species complex. Based on herbarium specimens, we hypothesize that these three groups in the P. maydis species complex originated from Central America, Mexico, and the Caribbean. Although two of these groups were only found on maize, the third and largest group contained contemporary strains found on maize and other grass hosts, as well as herbarium specimens from maize and other grasses that include 10 species of Phyllachora. The herbarium specimens were previously identified based on morphology and host association. This work represents the first attempt at molecular characterization of Phyllachora species infecting grass hosts and indicates some Phyllachora species can infect a broad range of host species and there may be significant synonymy in the Phyllachora genus.     

VEGF receptor 2/‐3 heterodimers detected in situ by proximity ligation on angiogenic sprouts

The vascular endothelial growth factors VEGFA and VEGFC are crucial regulators of vascular development. They exert their effects by dimerization and activation of the cognate receptors VEGFR2 and VEGFR3. Here, we have used in situ proximity ligation to detect receptor complexes in intact endothelial cells. We show that both VEGFA and VEGFC potently induce formation of VEGFR2/‐3 heterodimers. Receptor heterodimers were found in both developing blood vessels and immature lymphatic structures in embryoid bodies. We present evidence that heterodimers frequently localize to tip cell filopodia. Interestingly, in the presence of VEGFC, heterodimers were enriched in the leading tip cells as compared with trailing stalk cells of growing sprouts. Neutralization of VEGFR3 to prevent heterodimer formation in response to VEGFA decreased the extent of angiogenic sprouting. We conclude that VEGFR2/‐3 heterodimers on angiogenic sprouts induced by VEGFA or VEGFC may serve to positively regulate angiogenic sprouting.     

An Introduction to a Special Issue on Large‐Scale Submerged Aquatic Vegetation Restoration Research in the Chesapeake Bay: 2003–2008

The Chesapeake Bay is one of the world's largest estuaries. Dramatic declines in the abundance and distribution of submerged aquatic vegetation (SAV) in the Chesapeake Bay over the last few decades led to a series of management decisions aimed at protecting and restoring SAV populations throughout the bay. In 2003, the Chesapeake Bay Program established a goal of planting 405 ha of SAV by 2008. Realizing that such an ambitious goal would require the development of large‐scale approaches to SAV restoration, a comprehensive research effort was organized, involving federal and state agencies, academia, and the private sector. This effort differs from most other SAV restoration programs due to a strong emphasis on the use of seeds rather than plants as planting stock, a decision based on the relatively low labor requirements of seeding. Much of the research has focused on the development of tools and techniques for using seeds in large‐scale SAV restoration. Since this research initiative began, an average of 13.4 ha/year of SAV has been planted in the Chesapeake Bay, compared to an average rate of 3.6 ha/year during the previous 21 years (1983–2003). The costs of conducting these plantings are on a downward trend as the understanding of the limiting factors increases and as new advances are made in applied research and technology development. Although this effort was focused in the Chesapeake Bay region, the tools and techniques developed as part of this research should be widely applicable to SAV restoration efforts in other areas.     

Risky business: sexual and asexual reproduction in variable environments

Patterns of reproductive uncertainty can have an important influence on population dynamics. There is a crucial distinction between what we describe here as aggregate uncertainty (in which reproductive output in each generation is correlated among the individuals in a population) and idiosyncratic risk (in which reproductive output is independent across individuals). All else being equal, populations experiencing idiosyncratic risk enjoy a higher asymptotic growth rate than do those experiencing aggregate uncertainty. Therefore individuals in populations of the former type will have a competitive advantage over individuals in populations of the latter type. Applying this distinction to models of randomly fluctuating environments, we point out that genetic variation among offspring can serve to reduce aggregate uncertainty, transforming it into a more idiosyncratic form of risk. We show that this transformation underlies the dynamics observed in several previous models of the role of outcrossing in the evolution of sex.     

Design and synthesis of conformationally constrained 3-(N-alkylamino)propylphosphonic acids as potent agonists of sphingosine-1-phosphate (S1P) receptors

A series of conformationally constrained 3-(N-alkylamino)propylphosphonic acids were systematically synthesized and their activities as S1P receptor agonists were evaluated. Several pyrrolidine and cyclohexane analogs had S1P receptor profiles comparable to the acyclic lead compound, 3-(N-tetradecylamino)propylphosphonic acid (3), lowered circulating lymphocytes in mice after iv administration and were thus identified as being suitable for further investigations.     

The discovery of 3-(N-alkyl)aminopropylphosphonic acids as potent S1P receptor agonists

3-(N-Alkyl)aminopropylphosphonic acids are potent agonists of four of the five known sphingosine-1-phosphate (S1P) receptor subtypes.