Background: Insulin-like growth factor-I (IGF-I), essential for normal human growth in utero and postnatally, mediates its effects through the IGF-I receptor (IGF1R), a widely expressed, cell-surface tyrosine kinase receptor. Five cases of heterozygous mutations in the IGF1R gene have been identified in patients with varying degrees of intrauterine and postnatal growth retardation. Objective: Analysis of the IGF1R gene in a short-statured patient and his affected family members. Patient: The male patient, with a height of -3.1 SDS (age of 12 years), had normal circulating levels of GHBP, IGF-I and IGFBP-3. His mother (-4.6 SDS), one of his siblings (-1.94 SDS), and several other maternal family members were also short-statured. Results: The patient, his mother and the short-statured sibling carry a novel heterozygous 19-nucleotide duplication within exon 18 of the IGF1R gene, which introduces a premature termination codon at codon 1106 of the IGF1R open reading frame on one allele. Analyses of the primary dermal fibroblasts derived from the patient and family members indicated that the IGF1R mRNA expressed from the mutant allele was degraded through the Nonsense-Mediated mRNA Decay (NMD) pathway, resulting in reduced amount of wild-type IGF1R protein, and subsequently, diminished activation of the IGF1R pathway. Conclusions: The mutation results in haploinsufficiency of IGF1R protein due to Nonsense-Mediated mRNA Decay, and is associated with familial short stature.

With Rumex K-1 seedlings as test materials, this paper studied the effects of different concentration (100-300 mmol x L(-1)) NaCl and KCl on their leaf photosynthetic activity and osmotic adjustment. The results showed that at the concentration of 200 mmol x L(-1), NaCl had greater inhibition effect on the leaf photosynthetic activity than KCl, but at 300 mmol x L(-1), the inhibition effect of KCl was much greater than NaCl. After treated with 300 mmol x L(-1) of KCl and NaCl, the leaf water potential was -0.93 MPa and -1.05 MPa, and the osmotic potential was -1.43 MPa and -1.10 MPa, respectively, indicating that the increased damage caused by 300 mmol x L(-1) of KCl was not from osmotic stress. Under the stress of 300 mmol KCl x L(-1), the leaf Na+ concentration decreased by 88.6%, compared with the control, while the supplement of 25 mmol NaCl x L(-1) could obviously alleviate the damage of KCl on leaf photosynthesis, which proved that the deficit of Na+ and the accumulation of K+ in Rumex K-1 leaves could be responsible to the enhanced damage caused by 300 mmol x L(-1) of KCl.

The great Sumatra-Andaman earthquake and tsunami of 2004 was a dramatic reminder of the importance of understanding the seismic and tsunami hazards of subduction zones. In March 2005, the Sunda megathrust ruptured again, producing an event of moment magnitude (M(w)) 8.6 south of the 2004 rupture area, which was the site of a similar event in 1861 (ref. 6). Concern was then focused on the Mentawai area, where large earthquakes had occurred in 1797 (M(w)= 8.8) and 1833 (M(w)= 9.0). Two earthquakes, one of M(w)= 8.4 and, twelve hours later, one of M(w)= 7.9, indeed occurred there on 12 September 2007. Here we show that these earthquakes ruptured only a fraction of the area ruptured in 1833 and consist of distinct asperities within a patch of the megathrust that had remained locked in the interseismic period. This indicates that the same portion of a megathrust can rupture in different patterns depending on whether asperities break as isolated seismic events or cooperate to produce a larger rupture. This variability probably arises from the influence of non-permanent barriers, zones with locally lower pre-stress due to the past earthquakes. The stress state of the portion of the Sunda megathrust that had ruptured in 1833 and 1797 was probably not adequate for the development of a single large rupture in 2007. The moment released in 2007 amounts to only a fraction both of that released in 1833 and of the deficit of moment that had accumulated as a result of interseismic strain since 1833. The potential for a large megathrust event in the Mentawai area thus remains large.

Context: Among over 250 cases of GH insensitivity syndrome (GHIS) reported to date, the largest cohort was identified in southern Ecuador. In the Ecuadorian GHIS cohort, a sense mutation (GAA>GAG) at codon E180 of GH receptor (GHR(E180sp)), results in deletion of codons 181-188. No functional studies of this mutation have been performed, nor have different mutations at codon 180 been reported. Objective: We now report identification of a novel GHR mutation, also within codon E180, in two distantly related GHIS subjects of Inuit origin, and provide mechanistic insights into the defects caused by the Inuit and Ecuadorian GHR mutations. Patients: The two Inuit subjects, with heights of -5 SD score (SDS) and -7 SDS, respectively, had elevated circulating levels of GH, but low levels of GH-binding protein, IGF-I, and IGF-binding protein-3. Results: Both Inuit subjects carry the same novel nonsense homozygous GHR mutation at codon E180 (GAA->TAA, E180X). In vitro reconstitution experiments demonstrated that GHR(E180sp), but not GHR(E180X), could be stably expressed. GHR(E180sp), however, could not bind GH and could neither activate STAT5b nor induce STAT5b-dependent gene expression upon GH treatment. Furthermore, the GHR(E180sp), which has a deletion of eight amino acid residues within the GHR dimerization domain, while retaining the ability to homodimerize, was defective in trafficking to the cell surface. Conclusions: The E180X mutation identified in two Inuit patients resulted in a truncated, unstably expressed GHR variant, whereas the E180 splicing mutation previously identified in the Ecuadorian cohort, affected both GH-binding and GHR-trafficking, and rendered the abnormal GHR nonfunctional.

OBJECTIVE: Insulin-like growth factor binding protein-3 (IGFBP-3) has been shown to exhibit diverse biological actions, including IGF-independent effects on cell growth and cell death. Here we report that IGFBP-3 sensitizes prostate cancer cells to interferon-gamma (IFN-gamma)-induced apoptosis and inhibition of cell proliferation. DESIGN: The cell growth or cell death of prostate cells in response to the treatments of IGFBPs and/or IFN-gamma was measured, and the signaling pathways mediating these actions assessed. RESULTS: Cell proliferation was minimally affected when M12 prostate cancer cells were treated with exogenous IGFBP-3 (1-5mug/ml), IGFBP-1 (1-5mug/ml) or IFN-gamma (20U/ml). However, strong inhibition of cell growth and significant apoptosis were observed when M12 cells were co-treated with IGFBP-3 and IFN-gamma, but not with IGFBP-1 and IFN-gamma. These effects were IGF-independent and appear not to require intracellular localization of IGFBP-3, as similar results were obtained with mutants of IGFBP-3 that either could not bind IGF or has impaired ability to be internalized. Further analyses revealed that IGFBP-3, but not IGFBP-1, could significantly enhance the weak tyrosine phosphorylation of STAT1 induced by IFN-gamma (20U/ml) alone. The IGFBP-3-promoted apoptosis in the presence of IFN-gamma could also be abrogated by blockade of the mTOR pathway with its pharmacological inhibitors, LY294002 or rapamycin. CONCLUSIONS: These results demonstrated that in a cancer cell line not responsive to exogenous IGFBP-3 alone, IGFBP-3 sensitized the cells to the anti-proliferative, proapoptotic actions of IFN-gamma through an IGF-independent, STAT1- and mTOR-dependent mechanism.

Context: Primary Growth Hormone Insensitivity (GHI) or Laron Syndrome, caused by mutations of the GH receptor (GHR) gene, has a clinical phenotype of postnatal growth failure associated with normal-elevated serum concentrations of GH and low serum levels of IGF-I. Objective: We investigated the clinical and biochemical implications of molecular defects in the GHR gene in an Austrian family with two daughters who were GHI. Patients: Patient 1 (height,-4.8 SD score (SDS)) and patient 2 (height,-5.0 SDS) had elevated circulating levels of GH, low-normal levels of GH-binding protein, and abnormally low IGF-I (-5.0 SDS and -2.6 SDS respectively) and IGF-binding protein-3 (-2.6 SDS and -2.0 SDS respectively). Results: Both patients carry novel compound, missense, heterozygous GHR mutations, C94S and H150Q. In vitro reconstitution experiments demonstrated that while each of the mutants could be stably expressed, GHR(C94S) lost its affinity for GH, and could neither activate STAT5b nor drive STAT5b-dependent gene transcription in response to GH (1-100 ng/ml). GHR(H150Q), showed normal affinity for GH, but impaired capacity for signal transduction. The compound heterozygote, and C94S heterozygote, but not the H150Q heterozygote, showed significant deficiency in activating GH-induced gene expression, corroborating diminished GH-induced STAT5b activation in fibroblasts carrying GHR(C94S), as either a compound heterozygote (in the patients) or as a simple heterozygote (in one parent). Conclusions: Each of the compound heterozygous mutations contributed additively to the pathological condition seen in the patients, and the more detrimental of the two mutations, C94S, may cause (partial) primary GHI, even in a heterozygous state.

We developed a system to noninvasively and repeatedly image in vivo hepatic GH signaling. GH regulates postnatal growth and metabolism. It affects numerous tissues, but has major effects in liver. We used nude mice for adenoviral-mediated delivery of a signal transducer and activator of transcription 5-dependent GH response element, a luciferase reporter to detect GH signaling pathway activation. We detected by noninvasive bioluminescence imaging GH-induced hepatic GH signaling serially within intact mice. Statistically significant effects of GH dose and time dependence were detected in the liver luciferase signal that peaked 3 h after GH injection. Codelivery of GH receptor significantly enhanced GH response, an effect that was further augmented by fasting. Our imaging system allows detailed in vivo analysis of GH signaling and action and may be a paradigm for studies of additional signaling pathways in liver and other tissues.

Context: A natural missense mutation in the signal transducer and activator of transcription 5b (STAT5b) gene was recently identified in association with a female patient presenting with severe growth failure and immune dysfunction. The mutation results in an alanine to proline substitution at residue 630 (A630P) in the src-homology-2 (SH2) domain, a region essential for docking of STATs to phospho-tyrosines on activated receptors, for STAT dimerization, and for stabilization of phospho-STAT-DNA interactions. Objective: The purpose of this study is to explore the molecular mechanisms underlying the Growth Hormone Insensitivity and IGF-I deficiency caused by the A630P mutated STAT5b. Results: In reconstitution experiments employing HEK293 cells, both growth hormone (GH) and interferon-gamma (IFNgamma) were unable to activate mutant STAT5b(A630P), as demonstrated by lack of immuno-detectable phospho-tyrosyl-STAT5b(A630P) and inability to drive luciferase-reporter activity. However, the Src family of non-receptor kinases (constitutively active v-src and EGF-induced c-src) tyrosine-phosphorylated STAT5b(A630P). The v-src-induced phospho-STAT5b(A630P) translocated to the nucleus, but, unlike wild-type Stat5b, was unable to bind DNA. Conclusions: The A630P mutation disrupts the SH2 architecture such that:(1) mutant STAT5b most likely cannot dock to phospho-tyrosines on ligand-activated receptors; and (2) stable interactions with DNA are prevented. Since STAT5b(A630P) is an inefficient signal transducer and transcription factor, the detrimental impact on signaling pathways important for normal growth and immunity explain, in part, the complex clinical phenotype of GH insensitivity and immune dysfunction.

Interferon-gamma (IFN-gamma) exhibits diverse biological activities, including control of cell growth and tumor suppression. Here, we report that the treatment of M12 cells, a human metastatic prostate cancer cell line, with IFN-gamma, resulted in marked inhibition of cell proliferation and induced apoptosis. These effects were not seen with either IFN-alpha or IFN-beta. M12 cells, like many other human cancer cells, contain constitutively activated signal transducer and activator of transcription 3 (STAT3). The basal levels of both Akt and ERK1/2 phosphorylation are also markedly elevated in M12 cells. Strikingly, IFN-gamma-induced apoptosis and growth inhibition of M12 cells were associated with persistent suppression of the constitutive tyrosine-phosphorylated STAT3 (pY-STAT3). The IFN-gamma-induced dephosphorylation of pY-STAT3, however, was inhibited when the mTOR pathway was specifically blocked by rapamycin. Inhibition of PI-3K with low-dose LY294002, or MAPK with PD98059 also suppressed the mTOR/p70 S6k pathway, and correlated with the blockage of IFN-gamma-induced dephosphorylation of pY-STAT3. Simultaneously, treatment with LY294002, PD98059, or rapamycin abolished IFN-gamma-induced apoptosis in M12 cells. The inhibition of the mTOR pathway, however, did not affect IFN-gamma-induced activation of STAT1 pathway, and suppression of STAT1 expression by siRNA had no effect on IFN-gamma-induced dephosphorylation of pY-STAT3. Taken together, these results demonstrate that an intact mTOR pathway is critical for IFN-gamma-induced suppression of pY-STAT3 and apoptosis. Our study thus provides novel insights into the contributions of signaling pathways other than the classical JAK/STAT1 pathway in the anti-proliferative, proapoptotic actions of IFN-gamma.

The proposed insulin-like growth factor binding protein (IGFBP) superfamily are a group of secreted proteins that are structurally, functionally and evolutionarily related, and include six IGFBPs and over 10 IGFBP-related proteins. The IGFBPs have high affinities for insulin-like growth factors (IGFs), thereby modulating the mitogenic, anti-apoptotic and metabolic actions of IGFs. In addition, IGFBPs, particularly IGFBP-3, also have IGF-independent, anti-proliferative and pro-apoptotic functions. The IGFBPs are, in turn, modulated by proteolysis. Epidemiological data correlating serum IGF/IGFBP levels with the risk of several human cancers suggest a possible protective role for IGFBP-3. In vitro studies suggest that the potential protective effects of IGFBP-3 involve both IGF-dependent and IGF-independent mechanisms. Further studies are necessary to demonstrate whether the ability of IGFBPs to inhibit the proliferation of cancer cells under in vitro conditions has significant clinical implications.

Polyploidization-induced genome variation in triticale (x Triticosecale Wittmack) was investigated using both AFLP and RFLP analyses. The AFLP analyses were implemented with both EcoRI-MseI (E-M) and PstI-MseI (P-M) primer combinations, which, because of their relative differences in sensitivity to cytosine methylation, primarily amplify repetitive and low-copy sequences, respectively. The results showed that the genomic sequences in triticale involved a great degree of variation including both repetitive and low-copy sequences. The frequency of losing parental bands was much higher than the frequency of gaining novel bands, suggesting that sequence elimination might be a major force causing genome variation in triticale. In all cases, variation in E-M primer-amplified parental bands was more frequent in triticale than that using P-M primers, suggesting that repetitive sequences were more involved in variation than low-copy sequences. The data also showed that the wheat (Triticum spp.) genomes were relatively highly conserved in triticales, especially in octoploid triticales, whereas the rye (Secale cereale L.) genome consistently demonstrated a very high level of genomic sequence variation (68%-72%) regardless of the triticale ploidy levels or primers used. In addition, when a parental AFLP band was present in both wheat and rye, the tendency of the AFLP band to be present in triticale was much higher than when it was present in only one of the progenitors. Furthermore, the cDNA-probed RFLP analyses showed that over 97% of the wheat coding sequences were maintained in triticale, whereas only about 61.6% of the rye coding sequences were maintained, suggesting that the rye genome variation in triticale also involved a high degree of rye coding sequence changes. The data also suggested that concerted evolution might occur in the genomic sequences of triticale. In addition, the observed genome variation in wheat-rye addition lines was similar to that in triticale, suggesting that wheat-rye addition lines can be used to thoroughly study the genome evolution of polyploid triticale.

The evolutionarily conserved fungal arginine attenuator peptide (AAP), as a nascent peptide, stalls the translating ribosome in response to the presence of a high concentration of the amino acid arginine. Here we examine whether the AAP maintains regulatory function in fungal, plant, and animal cell-free translation systems when placed as a domain near the N terminus or internally within a large polypeptide. Pulse-chase analyses of the radiolabeled polypeptides synthesized in these systems indicated that wild-type AAP functions at either position to stall polypeptide synthesis in response to arginine. Toeprint analyses performed to map the positions of stalled ribosomes on transcripts introduced into the fungal system revealed that ribosome stalling required translation of the AAP coding sequence. The positions of the stalled ribosomes were consistent with the sizes of the radiolabeled polypeptide intermediates. These findings demonstrate that an internal polypeptide domain in a nascent chain can regulate eukaryotic translational elongation in response to a small molecule. Apparently the peptide-sensing features are conserved in fungal, plant, and animal ribosomes. These data provide precedents for translational strategies that would allow domains within nascent polypeptide chains to modulate gene expression.

Neurospora crassa has 10 mapped supersuppressor (ssu) genes. In vivo studies indicate that they suppress amber (UAG) premature termination mutations but the spectrum of their functions remains to be elucidated. We examined seven ssu strains (ssu-1,-2,-3,-4,-5,-9, and -10) using cell-free translation extracts. We tested suppression by requiring it to produce firefly luciferase from a reading frame containing premature UAA, UGA, or UAG terminators. All mutants except ssu-3 suppressed UAG codons. Maximal UAG suppression ranged from 15% to 30% relative to controls containing sense codons at the corresponding position. Production from constructs containing UAA or UGA was 1-2%, similar to levels observed with all nonsense codons in wild-type and ssu-3 extracts. UAG suppression was also seen using [35S]Met to radiolabel polypeptides. Suppression enabled ribosomes to continue translation elongation as determined using the toeprint assay. tRNA from supersuppressors showed suppressor activity when added to wild-type extracts. Thus, these supersuppressors produce amber suppressor tRNA.

The ability to map the position of ribosomes and their associated factors on mRNAs is critical for an understanding of translation mechanisms. Earlier approaches to monitoring these important cellular events characterized nucleotide sequences rendered nuclease-resistant by ribosome binding. While these approaches furthered our understanding of translation initiation and ribosome pausing, the pertinent techniques were technically challenging and not widely applied. Here we describe an alternative assay for determining the mRNA sites at which ribosomes or other factors are bound. This approach uses primer extension inhibition, or "toeprinting," to map the 3' boundaries of mRNA-associated complexes. This methodology, previously used to characterize initiation mechanisms in prokaryotic and eukaryotic systems, is used here to gain an understanding of two interesting translational regulatory phenomena in the fungi Neurospora crassa and Saccharomyces cerevisiae:(a) regulation of translation in response to arginine concentration by an evolutionarily conserved upstream open reading frame, and (b) atypical termination events that occur as a consequence of the presence of premature stop codons.