Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity.

To identify structural characteristics of the closely related cell surface receptors for insulin and IGF-I that define their distinct physiological roles, we determined the complete primary structure of the human IGF-I receptor from cloned cDNA. The deduced sequence predicts a 1367 amino acid receptor precursor, including a 30-residue signal peptide, which is removed during translocation of the nascent polypeptide chain. The 1337 residue, unmodified proreceptor polypeptide has a predicted Mr of 151,869, which compares with the 180,000 Mr IGF-I receptor precursor. In analogy with the 152,784 Mr insulin receptor precursor, cleavage of the Arg-Lys-Arg-Arg sequence at position 707 of the IGF-I receptor precursor will generate alpha (80,423 Mr) and beta (70,866 Mr) subunits, which compare with approximately 135,000 Mr (alpha) and 90,000 Mr (beta) fully glycosylated subunits.     

A map of 22 loci on human chromosome 22.

We constructed a genetic linkage map of the entire long arm of human chromosome 22 with 30 polymorphic markers, defining 22 loci. The map consists of a continuous linkage group 110 cM long, when male and female recombination fractions are combined; average distance between the loci is 5.2 cM. All loci were placed on the map with high support against alternative orders (odds in excess of 1000:1). The order of loci presented in our map is in full agreement with that of the previous linkage maps of chromosome 22 and with the physical assignment of markers. Two markers included in this map, KI-831 (D22S212) and pEFZ31 (D22S32), allowed us to better define the region of the (11;22) translocation breakpoint specific for Ewing sarcoma. Ten additional polymorphic markers were placed on the 22-loci map with odds lower than 1000:1 against alternative locations. In total, we have introduced 29 new markers on the linkage map of chromosome 22.     

Allele-Specific PCR with Competitive Probe Blocking for Sensitive and Specific Detection of BRAF V600E in Thyroid Fine-Needle Aspiration Specimens

Objective: To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR). Study Design: Tumor-enriched DNA was extracted from FNA smears, formalin-fixed paraffin-embedded (FFPE) sections, or NR specimens from 37 patients with confirmed papillary thyroid carcinoma or benign findings. An allele-specific primer selectively amplified the 1799 T>A BRAF mutation while simultaneously blocking amplification of wild-type (WT) BRAF with an unlabeled probe during PCR. Mutation detection was accomplished by melting analysis of the probe. Results: Allele-specific/blocking probe PCR confirmed the BRAF mutation status for 20 of 24 paired FNA/FFPE samples previously tested by fluorescent probe real-time PCR. For the other 4 cases, the sensitive PCR method detected the BRAF mutation in all paired FNA/FFPE samples. Previously, the mutation had been detected in only the FFPE samples. The BRAF mutation was also detected in some NR specimens. Conclusion: Treatment of patients with thyroid nodules is guided by FNA biopsy, which can be scantly cellular, necessitating a sensitive test that can detect low levels of BRAF V600E mutation in a WT background. We report increased detection of BRAF V600E in FNA specimens using allele-specific/blocking probe PCR, which has an analytical sensitivity of 0.01%.     

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl⁻ and Na⁺ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%–65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.     

Loss of a large grazer impacts savanna grassland plant communities similarly in North America and South Africa

Large herbivore grazing is a widespread disturbance in mesic savanna grasslands which increases herbaceous plant community richness and diversity. However, humans are modifying the impacts of grazing on these ecosystems by removing grazers. A more general understanding of how grazer loss will impact these ecosystems is hampered by differences in the diversity of large herbivore assemblages among savanna grasslands, which can affect the way that grazing influences plant communities. To avoid this we used two unique enclosures each containing a single, functionally similar large herbivore species. Specifically, we studied a bison (Bos bison) enclosure at Konza Prairie Biological Station, USA and an African buffalo (Syncerus caffer) enclosure in Kruger National Park, South Africa. Within these enclosures we erected exclosures in annually burned and unburned sites to determine how grazer loss would impact herbaceous plant communities, while controlling for potential fire-grazing interactions. At both sites, removal of the only grazer decreased grass and forb richness, evenness and diversity, over time. However, in Kruger these changes only occurred with burning. At both sites, changes in plant communities were driven by increased dominance with herbivore exclusion. At Konza, this was caused by increased abundance of one grass species, Andropogon gerardii, while at Kruger, three grasses, Themeda triandra, Panicum coloratum, and Digitaria eriantha increased in abundance.     

Rewiring bacteria, two components at a time

In this issue, Skerker et al. (2008) present a rational method for rewiring the protein-protein interactions and output responses of prokaryotic two-component signal transduction systems. This work has important implications for understanding the specificity of protein interactions and for designing protein-based synthetic signaling cascades.     

Disturbance Frequency and Community Stability in Native Tallgrass Prairie

Ecological communities are spatially and temporally variable in response to a variety of biotic and abiotic forces. It is not always clear, however, if spatial and temporal variability leads to instability in communities. Instability may result from strong biotic interactions or from stochastic processes acting on small populations. I used 10-15 yr of annual data from the Konza Prairie Long-Term Ecological Research site to examine whether plant, breeding bird, grasshopper, and small mammal communities in tallgrass prairie exhibit stability or directional change in response to different experimentally induced fire frequencies. Based on ordination and ANOVA, plant and grasshopper communities on annually burned sites differed significantly from plant and grasshopper communities on less frequently burned sites. Breeding birds and small mammals differed among sites as well, but these differences were not clearly related to disturbance frequency. A modified time series analysis indicated that plant communities were undergoing directional change (unstable) on all watersheds, regardless of fire frequency. Contrary to expectations, directional change was greatest on the annually burned sites and lowest on the infrequently burned sites. Unlike the plant communities, breeding bird, grasshopper, and small mammal communities were temporally stable, despite high-compositional variability from 1 yr to the next. Stability among the consumer communities within these dynamic plant communities occurs because three-dimensional vegetation structure does not change over time, despite changes in plant species composition. Evidence suggests that instability in the plant community results from strong biotic interactions among temporally persistent core species and stochastic dynamics among infrequent satellite species. Overall, community stability cannot be assessed if the pattern of temporal dynamics is unknown. Long-term empirical studies of different taxa under different disturbance regimes are needed to determine over what time frames and spatial scales communities may be stable. Such studies are essential for the development of generalities regarding the relationship between disturbance frequency and community stability in terrestrial and aquatic systems.     

The JNK-interacting protein-1 scaffold protein targets MAPK phosphatase-7 to dephosphorylate JNK

The c-Jun N-terminal kinase (JNK) group of mitogen-activated protein kinases (MAPKs) are activated by pleiotropic signals including environmental stresses, growth factors, and hormones. A subset of JNK can bind to distinct scaffold proteins that also bind upstream kinases of the JNK pathway, allowing sequential kinase activation within a signaling module. The JNK-interacting protein-1 (JIP-1) scaffold protein specifically binds JNK, MAP kinase kinase 7, and members of the MLK family and is essential for stress-mediated JNK activation in neurones. Here we report that JIP-1 also binds the dual-specificity phosphatases MKP7 and M3/6 via a region independent of its JNK binding domain. The C-terminal region of MKP7, homologous to that of M3/6 but not other DSPs, is required for interaction with JIP-1. When MKP7 is bound to JIP-1 it reduces JNK activation leading to reduced phosphorylation of the JNK target c-Jun. These results indicate that the JIP-1 scaffold protein modulates JNK signaling via association with both protein kinases and protein phosphatases that target JNK.     

Remodeling of synaptic actin induced by photoconductive stimulation.

Use-dependent synapse remodeling is thought to provide a cellular mechanism for encoding durable memories, yet whether activity triggers an actual structural change has remained controversial. We use photoconductive stimulation to demonstrate activity-dependent morphological synaptic plasticity by video imaging of GFP-actin at individual synapses. A single tetanus transiently moves presynaptic actin toward and postsynaptic actin away from the synaptic junction. Repetitive spaced tetani induce glutamate receptor-dependent stable restructuring of synapses. Presynaptic actin redistributes and forms new puncta that label for an active synapse marker FM5-95 within 2 hr. Postsynaptic actin sprouts projections toward the new presynaptic actin puncta, resembling the axon-dendrite interaction during synaptogenesis. Our results indicate that activity-dependent presynaptic structural plasticity facilitates the formation of new active presynaptic terminals.