Short-term effects of anastrozole treatment on insulin-like growth factor system in postmenopausal advanced breast cancer patients

Insulin-like growth factors (IGFs) play a fundamental role in cancer development by acting in both an endocrinal and paracrinal manner, and hormone breast cancer treatments affect the IGF system by modifying circulating growth factor levels. We evaluated total IGF-1, IGF-2, IGF binding protein (IGFBP)-1 and IGFBP-3 in the blood of 34 postmenopausal advanced breast cancer patients (median age 63 years, range 41–85) treated with anastrozole, a non-steroidal structure aromatase inhibitor (NSS-AI). The plasma samples were obtained at baseline, and after 2, 4, 8 and 12 weeks of treatment. The IGFs were quantitated by means of sensitive radioimmunoassays (RIAs). IGF-1 significantly increased during anastrozole treatment (baseline versus 12 weeks, P=0.031), IGF-2 showed a trend towards an increase, and IGFBP-1 constantly but not significantly decreased; IGFBP-3 did not seem to be affected at all. The anastrozole-induced changes in IGFs and IGFBP-1 appeared to be different in the patients receiving a clinical benefit from those observed in non-responders. We have previously shown that letrozole (a different type of NSS-AI) modifies blood IGF-1 levels, and the results of this study of the biological effects of anastrozole on the components of the IGF system confirm our previous observations.     

Behaviour of ribosomal genes and nucleolar domains during activation in sugarcane (Saccharum officinarum L.) root primordia: from the unsoaked quiescent state to the steady state of proliferation

Changes in the organisation of ribosomal genes and nucleolar protein components were analysed in sugarcane (Saccharum officinarum L. cv Cristalina) from the time the quiescent primordia of the radical bands of nodes were stimulated to proliferate by water imbibition, until the meristematic population reached the steady state of proliferation in the growing roots. The kinetics of proliferation was evaluated by flow cytometry, and by the mitotic indexes, in roots of different lengths. All the quiescent cells were in a pre-replicative state (G0), with a 2C DNA content. During their activation process, they progressively reached the steady state of proliferation (mitotic index 7%), with rather fixed frequencies for cells with 2C (G1), 4C (G2), and values between them corresponding to cells replicating their DNA. Decondensation of the ribosomal genes was followed by FISH with probes for the major 25S and 18S rRNAs, and variations in the numbers of nucleoli were recorded in squashed cells after silver staining. The ultrastructure of nucleoli was analysed by electron microscopy, using the EDTA regressive staining for ribonucleoproteins. Quiescent nucleoli showed a clear segregation of their main components: Fibrillar Centre, Dense Fibrillar Component and Cajal's bodies while lacked any Granular Component. However the proliferating ones showed them highly intermingled, except for the Cajal's bodies. Our results revealed a high plasticity of the nucleolar domains in response to cell activation, and allowed to establish a correlation between dispersion of NORs with formation of small fibrillar centers and a nucleolus with all its domains intermingled, and the activation of cell proliferation during root sprouting.     

Calculation of hydrodynamic properties of small nucleic acids from their atomic structure

Hydrodynamic properties (translational diffusion, sedimentation coefficients and correlation times) of short B-DNA oligonucleotides are calculated from the atomic-level structure using a bead modeling procedure in which each non-hydrogen atom is represented by a bead. Using available experimental data of hydrodynamic properties for several oligonucleotides, the best fit for the hydrodynamic radius of the atoms is found to be ~2.8 Å. Using this value, the predictions for the properties corresponding to translational motion and end-over-end rotation are accurate to within a few percent error. Analysis of NMR correlation times requires accounting for the internal flexibility of the double helix, and allows an estimation of ~0.85 for the Lipari–Szabo generalized order parameter. Also, the degree of hydration can be determined from hydrodynamics, with a result of ~0.3 g (water)/g (DNA). These numerical results are quite similar to those found for globular proteins. If the hydrodynamic model for the short DNA is simply a cylindrical rod, the predictions for overall translation and rotation are slightly worse, but the NMR correlation times and the degree of hydration, which depend more on the cross-sectional structure, are more severely affected.     

Properties of triple helices formed by parallel-stranded hairpins containing 8-aminopurines

Parallel-stranded hairpins with a polypyrimidine sequence linked to a complementary purine carrying 8-aminopurines such as 8-aminoadenine, 8-aminoguanine and 8-aminohypoxanthine bind polypyrimidine sequences complementary (in an antiparallel sense) to the purine part by a triple helix. The relative stabilities of triplexes were assessed by UV-absorption melting experiments as a function of pH and salt concentration. Hairpins carrying 8-aminopurines give very stable triple helical structures even at neutral pH, as confirmed by gel-shift experiments, circular dichroism and nuclear magnetic resonance spectroscopy. The modified hairpins may be redesigned to cope with small interruptions in the polypyrimidine target sequence.     

Challenges for mass production of nematodes in submerged culture

Nematodes of Steinernema and Heterorhabditis genera are used as agents in insect biocontrol programs. They are associated with specific bacteria which are also involved in the mechanism of pathogenicity and which are consumed by nematodes as living food. S. feltiae has various developmental stages in its life cycle, including four juvenile stages, adults and the free living form. During mating, males coil themselves around the female, which is around 1 cm long. Successful commercialization of nematode-bacteria biocontrol products depends on the ability to produce sufficient quantities of these products at competitive prices for a full pest control program. This could be feasible if high cell density submerged cultures are designed and implemented; however, major problems related to nematodes mass production in a bioreactor remain unsolved due to the lack of knowledge about the physiological aspects of the nematode, bacteria and nematode-bacteria association, interaction between the three phases present in the bioreactor (liquid, gas, nematodes-bacteria), possibility of mating under hydrodynamic stress conditions, etc. We have found that the two most important engineering aspects to take into account the mass propagation of nematodes are oxygen transfer rate and hydrodynamics to allow mating and to avoid mechanical damage of juveniles in stage 2.This article focuses on several aspects related to the fermentation system such as kinetics of growth, shear stress, hydrodynamics fields in the bioreactor and oxygen demand. Also, results published by other groups, together with those of our own, will be discussed in relation to the main challenges found during the fermentation process.     

Aesthetic eyelid ptosis correction: a review of technique and cases

Upper eyelid ptosis can present both functional and aesthetic problems. Because proper correction of ptosis can be difficult to achieve, numerous surgical procedures have been developed. Plication of levator aponeurosis can be combined with aesthetic blepharoplasty and facial rejuvenation procedures to successfully address ptosis. The authors assessed the effectiveness of levator aponeurosis plication for correction of acquired upper eyelid ptosis in patients presenting for concomitant cosmetic facial procedures. The medical records of 74 consecutive patients (68 women and six men) who had upper eyelid ptosis correction in conjunction with cosmetic facial procedures from January of 1994 to January of 2000 were reviewed. During this period, 400 endoscopic forehead lifts and 479 face lifts were performed. The correction was performed through an external upper blepharoplasty approach removing an ellipse of skin and orbicularis muscle. Once the orbital septum was opened, a plication of the levator aponeurosis was accomplished by one or more horizontal mattress sutures of 6-0 clear nylon (with the first bite placed at or just medial to the vertical level of the pupil). The average follow-up period was 14 months. Long-term correction of the ptosis was excellent. The complications were minor, with the most common occurrence being asymmetry. Revisions were performed on only four patients. Correction of ptosis can be performed safely and effectively in conjunction with periorbital and facial rejuvenation. The technique described is simple, reliable, and reproducible.     

Development and validation of a gas chromatography-mass spectrometry assay for hair analysis of amphetamine, methamphetamine and methylenedioxy derivatives

A procedure based on gas chromatography-mass spectrometry (GC-MS) is described for the determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA, ecstasy), 3,4-methylenedioxyethylamphetamine (MDE or MDEA) and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in hair. Hair samples were digested with 1 M sodium sulfide at 37 degrees C (by shaking for 3 h and was kept at room temperature overnight), and extracted with two sequential extraction procedures: liquid-liquid extraction with tert-butyl methyl ether and solid-phase extraction with Bond-Elut Certify columns. Extracted analytes were derivatised with N-methyl-bis(trifluoroacetamide), separated by a 5% phenylmethylsilicone column and determined by a mass spectrometer detector in selected ion monitoring mode. A good reproducibility (intra-assay R.S.D.=1.5-15.7%), accuracy (intra-assay error = 2.0-11.7%) and sensitivity (LOD=0.03-0.08 ng/mg hair) were attained. The method was successfully applied to the analysis of the proximal (1 cm) hair segment to assess recent self-reported use in "ecstasy" consumers. Otherwise, further studies are needed to validate methodology developed in case of amphetamine consumption.     

Estimating domain orientation of two human antibody IgG4 chimeras by crystallohydrodynamics

A modified crystallohydrodynamic approach introduced in 2001 is applied to two human IgG4 constructs from mouse IgG1. The constructs were point mutants of the chimeric antibody molecule cB72.3(4): cB72.3(4A), devoid of inter-chain disulfide bridging, and cB72.3(4P), which has full inter-chain bridging. As before, the known crystallographic structures for the Fab and Fc domains were combined with the measured translational frictional ratios to obtain an estimate for the apparent time-averaged hydration of the domains and hence for that of the intact molecule. The original approach was modified with the hydrated dimensions of the domains being applied, rather than the anhydrous crystallographic dimensions, for assessing the inter-domain orientations using the algorithms HYDROSUB and SOLPRO. Both chimeric IgG4 molecules were found to have open, rather than compact, structures, in agreement with the previous study on wild-type human IgG4. The insertion of a frictionless connector between the domains was necessary, however, for representing the cB72.3(4A) chimera. It therefore appears that the inter-chain disulfide bonds act as physical constraints in the cB72.3(4P) chimera, forcing the antibody domains together and producing a less elongated structure than that of cB72.3(4A). The open structures produced for the two IgG4 chimeras showed similarity to those structures identified for murine IgG1 and IgG2a molecules through X-ray crystallography.Presented at the conference for Advances in Analytical Ultracentrifugation and Hydrodynamics, 8–11 June 2002, Grenoble, France     

Human syndromes with genomic instability and multiprotein machines that repair DNA double-strand breaks

The present report deals with the functional relationships among protein complexes which, when mutated, are responsible for four human syndromes displaying cancer proneness, and whose cells are deficient in DNA double-strand break (DSB) repair. In some of them, the cells are also unable to activate the proper checkpoint, while in the others an unduly override of the checkpoint-induced arrest occurs. As a consequence, all these patients display genome instability. In ataxia-telangiectasia, the mutated protein (ATM) is a kinase, which acts as a transducer of DNA damage signalling. The defective protein in the ataxia-telangiectasia-like disorder is a DNase (the Mre11 nuclease) that in vivo produces single-strand tails at both sides of DSBs. Mre11 is always present with the Rad50 ATPase in a protein machine: the nuclease complex. In mammals, this complex also contains nibrin, the protein mutated in the Nijmegen syndrome. Nibrin confers new abilities to the nuclease complex, and can also bind to BRCA1 (one of the two proteins mutated in familial breast cancer). BRCA1 has a central motif that binds with high affinity to cruciform DNA, a structure present in places where the DNA loops are anchored to the chromosomal axis or scaffold. The BRCA1 x cruciform DNA complex should be released to allow the nuclease complex to work in DNA recombinational repair of DSBs. BRCA1 also acts as a scaffold for the assembly of ATPases such as Rad51, responsible for the somatic homologous recombination. Loss of the BRCA1 gene prevents cell survival after exposure to cross-linkers. The BRCA1-RING domain is an E3-ubiquitin ligase. It can mono-ubiquitinate the FANCD2 protein, mutated in one of the Fanconi anemia complementation groups, to regulate it. Finally, during DNA replication, the nuclease complex and its activating ATM kinase are integrated in the BRCA1-associated surveillance complex (BASC) that contains, among others, enzymes required for mismatch excision repair. In short, the proteins missing in these syndromes have in common their BRCA1-mediated assembly into multimeric machines responsible for the surveillance of DNA replication, DSB recombinational repair, and the removal of DNA cross-links.     

Disruption of the endocytic protein HIP1 results in neurological deficits and decreased AMPA receptor trafficking

Huntingtin interacting protein 1 (HIP1) is a recently identified component of clathrin-coated vesicles that plays a role in clathrin-mediated endocytosis. To explore the normal function of HIP1 in vivo, we created mice with targeted mutation in the HIP1 gene (HIP1(-/-)). HIP1(-/-) mice develop a neurological phenotype by 3 months of age manifest with a failure to thrive, tremor and a gait ataxia secondary to a rigid thoracolumbar kyphosis accompanied by decreased assembly of endocytic protein complexes on liposomal membranes. In primary hippocampal neurons, HIP1 colocalizes with GluR1-containing AMPA receptors and becomes concentrated in cell bodies following AMPA stimulation. Moreover, a profound dose-dependent defect in clathrin-mediated internalization of GluR1-containing AMPA receptors was observed in neurons from HIP1(-/-) mice. Together, these data provide strong evidence that HIP1 regulates AMPA receptor trafficking in the central nervous system through its function in clathrin-mediated endocytosis.