Defining the pathway to insulin-like growth factor system targeting in cancer

The insulin-like growth factors (IGEs; IGF-1 and IGF-2) play central roles in cell growth, differentiation, survival, transformation and metastasis. The biologic effects of the IGFs are mediated by the IGF-1 receptor (IGF-1R), a receptor tyrosine kinase with homology to the insulin receptor (IR). Dysregulation of the ICE system is well recognized as a key contributor to the progression of multiple cancers, with IGF-1R activation increasing the tumorigenic potential of breast, prostate, lung, colon and head and neck squamous cell carcinoma (HNSCC). Despite this relationship, targeting the IGF-1R has only recently undergone development as a molecular cancer therapeutic. As it has taken hold, we are witnessing a robust increase and interest in targeting the inhibition of IGF-1R signaling. This is accentuated by the list of over 30 drugs, including monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs) that are under evaluation as single agents or in combination therapies 1]. The ICE-binding proteins (IGFBPs) represent the third component of the ICE system consisting of a class of six soluble secretory proteins. They represent a unique class of naturally occurring ICE-antagonists that bind to and sequester IGF-1 and IGF-2, inhibiting their access to the IGF-1R. Due to their dual targeting of the IGFs without affecting insulin action, the IGFBPs are an untapped ``third'' class of IGF-1R inhibitors. in this commentary, we highlight some of the significant aspects of and prospects for targeting the IGF-1R and describe what the future may hold. (C) 2010 Elsevier Inc. All rights reserved.

Over-expression and purification strategies for recombinant multi-protein oligomers: A case study of Mycobacterium tuberculosis sigma/anti-sigma factor protein complexes

The function of a protein in a cell often involves coordinated interactions with one or several regulatory partners. It is thus imperative to characterize a protein both in isolation as well as in the context of its complex with an interacting partner. High resolution structural information determined by X-ray crystallography and Nuclear Magnetic Resonance offer the best route to characterize protein complexes. These techniques, however, require highly purified and homogenous protein samples at high concentration. This requirement often presents a major hurdle for structural studies. Here we present a strategy based on co-expression and co-purification to obtain recombinant multi-protein complexes in the quantity and concentration range that can enable hitherto intractable structural projects. The feasibility of this strategy was examined using the sigma factor/anti-sigma factor protein complexes from Mycobacterium tuberculosis. The approach was successful across a wide range of sigma factors and their cognate interacting partners. It thus appears likely that the analysis of these complexes based on variations in expression constructs and procedures for the purification and characterization of these recombinant protein samples would be widely applicable for other multi-protein systems. (C) 2010 Elsevier Inc. All rights reserved.

Use of adult male bonnet monkeys (Macaca radiata) for testing the biological activity of gonadotropin releasing hormone and its analogues

The effect of injecting agonistic and antagonistic analogues of gonadotropin releasing hormone analogues on serum testosterone levels was checked in adult and immature male bonnet monkeys. Of the agonistic analogues Buserelin, Ovurelin and D-Phe6 Gln8 GnRH were found to be most potent in increasing serum testosterone levels in the adult male bonnet monkeys. While 27-month-old monkeys responded well to des Gly10 GnRH, only marginal response was observed in the case of 15-month-old monkeys. Studies carried out with Ovurelin indicated that it was not effective in causing desensitization in adult monkeys. The antagonistic analogue was effective in blocking nocturnal surge of serum testosterone. Based on these studies it is suggested the adult male bonnet monkeys can be effectively used for testing the activity of GnRH analogues.

Involvement of cytoskeletal structures in nerve-growth-factor-mediated induction of ornithine decarboxylase.

Induction of ornithine decarboxylase elicited in response to nerve-growth factor in target organs is greatly decreased by preincubation of these tissues with cytoskeletal poisons such as vinblastine, diamide, cytochalasin B and colchicine. These results are interpreted as evidence for the involvement of receptor-associated cytoskeletal structures in mediating the nerve-growth-factor-specific induction of ornithine decarboxylase.

Involvement of cytoskeletal structures in nerve-growth-factor-mediated induction of ornithine decarboxylase

Induction of ornithine decarboxylase elicited in response to nerve-growth factor in target organs is greatly decreased by preincubation of these tissues with cytoskeletal poisons such as vinblastine, diamide, cytochalasin B and colchicine. These results are interpreted as evidence for the involvement of receptor-associated cytoskeletal structures in mediating the nerve-growth-factor-specific induction of ornithine decarboxylase.

Chronic suppression of testicular function by constant infusion of gonadotropin-releasing hormone agonist and testosterone supplementation in the bonnet monkey (Macaca radiata)

Objective: To study the efficacy of long-term buserelin acetate infusion to desensitize pituitary and block testicular function in adult male monkeys (Macaca radiata). Animals: Proven fertile male monkeys exhibiting normal testicular function. Protocol: Each of the control (n = 5) and experimental monkeys (n = 10) received a fresh miniosmotic pump every 21 days, whereas pumps in controls delivered vehicle of experimentals released 50-mu-g buserelin acetate every 24 hours. On day 170 (renewed every 60 days) a silastic capsule containing crystalline testosterone (T) was implanted in the experimental monkeys. At the end of 3 years, treatment was stopped, and recovery of testicular function and fertility monitored. Results: (1) Treatment resulted in marked reduction of nocturnal but not basal serum T; (2) the pituitary remained desensitized to buserelin acetate throughout the 3-year period; (3) animals were largely azoospermic with occasional oligospermia exhibited by two monkeys; and (4) withdrawal of treatment restored testicular function, with 70% of animals regaining fertility. Conclusion: Long-term infertility (but restorable) can be induced in male monkeys by constant infusion of buserelin acetate and T.

Effect of constant infusion of gonadotropin releasing hormone (GnRH) agonist Buserelin and antagonist CDB 2085 A using osmotic minipumps on testicular function in adult male bonnet monkey (Macaca radiata)

The effect of chronic infusion of gonadotropic hormone agonist Buserelin or antagonist CDB 2085 A for 15 weeks via alzet minipumps in adult male bonnet monkeys was studied. Infusion of Buserelin resulted in a decrease in the difference between serum testosterone values at 22.00 hours and 10.00 hours, decrease in responsiveness to injected Buserelin as judged by change in serum testosterone values from pre-injection values and decrease in sperm counts. Infusion of antagonist resulted in a decrease in the difference between serum testosterone values at 22.00 hours and 10.00 hours.

Dynamic Structure Factor of Sponge Phases

We predict the dynamic light scattering intensity S(q,t) for the L3 phase (anomalous isotropic phase) of dilute surfactant solutions. Our results are based on a Landau-Ginzburg approach, which was previously used to explain the observed static structure factor S(q, 0). In the extreme limit of small q, we find a monoexponential decay with marginal or irrelevant hydrodynamic interactions. In most other regimes the decay of S(q,t) is strongly nonexponential; in one case, it is purely algebraic at long times.

Cycloheximide enhances factor binding to the native 40S ribosomal subunit of Microsporum canis

Native and derived ribosomal particles from the mycelial cells of Microsporum canis grown in the presence and absence of cycloheximide were compared by CsCl equilibrium density gradient centrifugation. Since the buoyant densities of ribonucleoprotein complexes are dependent on the protein-RNA ratio, they reflect the composition of these particles. The native monosomes from cells grown in the presence and absence of cycloheximide had a buoyant density of 1.585 g/cc. The native 60S subunits showed a density of 1.540 g/cc from cells grown in both presence and absence of cycloheximide, while the derived subunits showed a density of 1.610 g/cc. The derived 40S subunits had a density of 1.550 g/cc while the native 40S showed a major species of density 1.535 g/cc with three other minor species ranging in densities from 1.450-1.390 g/cc. The mycelia grown in the presence of cycloheximide showed an increased proportion of native 40S subunits in the density range of 1.450-1.390 g/cc, indicating that the drug enhances factor binding to native ribosomal subunits in M. canis.

Higher twist and higher order contributions to the pion electromagnetic form factor

The O(m(pi)4/(m(u) + (d))2Q2) and O(alpha(S)2) corrections to the leading term of the perturbative QCD calculation of the pion electromagnetic form factor are examined numerically. Both sets of terms provide significant corrections for values of Q2 between 1 and 15 GeV2/c2.

The secretion of gonadotrophin-releasing hormone by peri-implantation embryos of the rhesus monkey: comparison with the secretion of chorionic gonadotrophin

Chorionic gonadotrophin (CG) is the first clear embryonic signal during early pregnancy in primates. CG has close structural and functional similarities to pituitary luteinizing hormone (LH) which is regulated by gonadotrophin releasing hormone (GnRH). To study the regulatory mechanism of CG secretion in primate embryos, we examined the production and timing of secretion of GnRH in peri-implantation embryos of the rhesus monkey. In-vivo fertilized/developed morulae and early blastocysts, recovered from non-superovulated, naturally-bred rhesus monkeys by non-surgical uterine flushing, were cultured in vitro to hatched, attached and post-attached blastocyst stages using a well-established culture system. We measured GnRH and CG in media samples from cultured embryos with a sensitive radioimmunoassay and bioassay, respectively. The secretion of GnRH (pg/ml; mean +/- SEM) by embryos (n = 20) commenced from low levels (0.32 +/- 0.05) during the pre-hatching blastocyst stage to 0.70 +/- 0.08 at 6-12 days and 1.30 +/- 0.23 at greater than or equal to 13 days of hatched blastocyst attachment and proliferation of trophoblast cells. GnRH concentrations in culture media obtained from embryos (n = 5) that failed to hatch and attach were mostly undetectable (less than or equal to 0.1). Samples that did not contain detectable GnRH failed to show detectable CG. Immunocytochemical studies, using a specific monoclonal anti-GnRH antibody (HU4H) as well as polyclonal antisera (LR-1), revealed that immunopositive GnRH cells were localized in pre-hatching blastocysts (n = 4), in blastocysts (n = 2) after 5-10 days of attachment and in monolayer cultures (n = 4) of well-established embryonic trophoblast cells. GnRH positive staining was seen only in cytotrophoblasts but not in syncytiotrophoblasts. Similarly, cytotrophoblast, but not syncytiotrophoblast, cells of the rhesus placenta were immunopositive. In controls, either in the absence of antibody or in the presence of antibody pre-absorbed with GnRH, these cells failed to show stain. These observations indicate, for the first time, that an immunoreactive GnRH is produced and secreted by blastocysts during the peri-attachment period and by embryo-derived cytotrophoblast cells in the rhesus monkey.

Implications of the recent high statistics determination of the pion electromagnetic form factor in the timelike region

The recently evaluated two-pion contribution to the muon g - 2 and the phase of the pion electromagnetic form factor in the elastic region, known from pi pi scattering by Fermi-Watson theorem, are exploited by analytic techniques for finding correlations between the coefficients of the Taylor expansion at t = 0 and the values of the form factor at several points in the spacelike region. We do not use specific parametrizations, and the results are fully independent of the unknown phase in the inelastic region. Using for instance, from recent determinations, < r(pi)(2)> = (0.435 +/- 0.005) fm(2) and F(-1.6 GeV2) = 0.243(-0.014)(+0.022), we obtain the allowed ranges 3.75 GeV-4 less than or similar to c less than or similar to 3.98 GeV-4 and 9.91 GeV-6 less than or similar to d less than or similar to 10.46 GeV-6 for the curvature and the next Taylor coefficient, with a strong correlation between them. We also predict a large region in the complex plane where the form factor cannot have zeros.

Molecular organization of the gene encoding Xenopus laevis transforming growth factor-beta 5

Transforming growth factor-beta s (TGF-beta 5) are multifunctional polypeptides, known to influence proliferation and differentiation of many cell types. TGF-beta 5 cDNA was cloned from Xenopus laevis and this isoform is unique to the amphibians. Here, we report the isolation and characterization of the TGF-beta 5 genomic clones to determine the structure of TGF-beta 5 gene. The gene consists of seven exons and all intron-exon boundaries follow the GT-AG consensus. The organization of TGF-beta 5 gene was identical to that of the mammalian TGF-beta isoforms, with the exception of position of the first splice junction. We determined the size of TGF-beta 5 gene to be approximately 20 kb.