Ligand migration in the apolar tunnel of Cerebratulus lacteus mini-hemoglobin

The large apolar tunnel traversing the mini-hemoglobin from Cerebratulus lacteus (CerHb) has been examined by x-ray crystallography, ligand binding kinetics, and molecular dynamic simulations. The addition of 10 atm of xenon causes loss of diffraction in wild-type (wt) CerHbO(2) crystals, but Leu-86(G12)Ala CerHbO(2), which has an increased tunnel volume, stably accommodates two discrete xenon atoms: one adjacent to Leu-86(G12) and another near Ala-55(E18). Molecular dynamics simulations of ligand migration in wt CerHb show a low energy pathway through the apolar tunnel when Leu or Ala, but not Phe or Trp, is present at the 86(G12) position. The addition of 10-15 atm of xenon to solutions of wt CerHbCO and L86A CerHbCO causes 2-3-fold increases in the fraction of geminate ligand recombination, indicating that the bound xenon blocks CO escape. This idea was confirmed by L86F and L86W mutations, which cause even larger increases in the fraction of geminate CO rebinding, 2-5-fold decreases in the bimolecular rate constants for ligand entry, and large increases in the computed energy barriers for ligand movement through the apolar tunnel. Both the addition of xenon to the L86A mutant and oxidation of wt CerHb heme iron cause the appearance of an out Gln-44(E7) conformer, in which the amide side chain points out toward the solvent and appears to lower the barrier for ligand escape through the E7 gate. However, the observed kinetics suggest little entry and escape (≤ 25%) through the E7 pathway, presumably because the in Gln-44(E7) conformer is thermodynamically favored.     

Contribution of social isolation, restraint, and hindlimb unloading to changes in hemodynamic parameters and motion activity in rats

The most accepted animal model for simulation of the physiological and morphological consequences of microgravity on the cardiovascular system is one of head-down hindlimb unloading. Experimental conditions surrounding this model include not only head-down tilting of rats, but also social and restraint stresses that have their own influences on cardiovascular system function. Here, we studied levels of spontaneous locomotor activity, blood pressure, and heart rate during 14 days under the following experimental conditions: cage control, social isolation in standard rat housing, social isolation in special cages for hindlimb unloading, horizontal attachment (restraint), and head-down hindlimb unloading. General activity and hemodynamic parameters were continuously monitored in conscious rats by telemetry. Heart rate and blood pressure were both evaluated during treadmill running to reveal cardiovascular deconditioning development as a result of unloading. The main findings of our work are that: social isolation and restraint induced persistent physical inactivity, while unloading in rats resulted in initial inactivity followed by normalization and increased locomotion after one week. Moreover, 14 days of hindlimb unloading showed significant elevation of blood pressure and slight elevation of heart rate. Hemodynamic changes in isolated and restrained rats largely reproduced the trends observed during unloading. Finally, we detected no augmentation of tachycardia during moderate exercise in rats after 14 days of unloading. Thus, we concluded that both social isolation and restraint, as an integral part of the model conditions, contribute essentially to cardiovascular reactions during head-down hindlimb unloading, compared to the little changes in the hydrostatic gradient.     

Policy on Synthetic Biology: Deliberation,Probability, and the Precautionary Paradox

Synthetic biology is a cutting‐edge area of research that holds the promise of unprecedented health benefits. However, in tandem with these large prospective benefits, synthetic biology projects entail a risk of catastrophic consequences whose severity may exceed that of most ordinary human undertakings. This is due to the peculiar nature of synthetic biology as a ‘threshold technology’ which opens doors to opportunities and applications that are essentially unpredictable. Fears about these potentially unstoppable consequences have led to declarations from civil society groups calling for the use of a precautionary principle to regulate the field. Moreover, the principle is prevalent in law and international agreements. Despite widespread political recognition of a need for caution, the precautionary principle has been extensively criticized as a guide for regulatory policy. We examine a central objection to the principle: that its application entails crippling inaction and incoherence, since whatever action one takes there is always a chance that some highly improbable cataclysm will occur. In response to this difficulty, which we call the ‘precautionary paradox,’ we outline a deliberative means for arriving at threshold of probability below which potential dangers can be disregarded. In addition, we describe a Bayesian mechanism with which to assign probabilities to harmful outcomes. We argue that these steps resolve the paradox. The rehabilitated PP can thus provide a viable policy option to confront the uncharted waters of synthetic biology research.     

Vulnerability to cavitation of leaf minor veins: any impact on leaf gas exchange?

Vulnerability to cavitation of leaf minor veins and stems of Laurus nobilis L. was quantified together with that of leaflets, rachides and stems of Ceratonia siliqua L. during air‐dehydration of 3‐year‐old branches. Embolism was estimated by counting ultrasound acoustic emissions (UAE) and relating them to leaf water potential (Ψ_L). The threshold Ψ_L for cavitation was less negative in L. nobilis than in C. siliqua according to the known higher drought resistance of the latter species. Leaf minor vein cavitation was also quantified by infiltrating leaves with fluorescein at different dehydration levels and observing them under microscope. Distinct decreases in the functional integrity of minor veins were observed during leaf dehydration, with high correlation between the two variables. The relationship between leaf conductance to water vapour (g_L) and Ψ_L showed that stomata of L. nobilis closed in response to stem and not to leaf cavitation. However, in C. siliqua, g_L decreased in coincidence to the leaf cavitation threshold, which was, nevertheless, very close to that of the stem. The hypothesis that stem cavitation acts as a signal for stomatal closure was confirmed, while the same role for leaf cavitation remains an open problem.     

Production and validation of the pharmacokinetics of a single-chain Fv fragment of the MGR6 antibody for targeting of tumors expressing HER-2

The HER-2 antigen, which is overexpressed in many breast carcinomas, is an ideal target for monoclonal antibodies due to its low expression in normal tissue and its homogeneous distribution in the tumor mass. We have developed and characterized the murine MAb MGR6 against HER-2, which is able to inhibit proliferation of tumor cells overexpressing HER-2. On the basis of these preclinical results, phase I studies in breast carcinoma patients were conducted and radiolocalization data indicated an antibody half life which directly paralleled that of other whole antibodies and thus resulting in a limited in vivo diagnostic capacity. To obtain a smaller reagent with possibly improved in vivo properties, a single chain variable fragment (scFv) of the original MGR6-producing hybridoma was generated by phage display technology. Biologically active MGR6 scFv was purified rapidly and at high yield by metal affinity chromatography. Competition FACS and ELISA analyses identified an epitope on the HER-2 extracellular domain that was shared by the scFv and the parental MAb. BIAcore analysis indicated a K_off of 9.3 × 10⁻⁴ s⁻¹, similar to that of the intact MGR6 MAb. Distribution and elimination half-lives of MGR6 scFv, calculated from in vivo preclinical evaluations, were much faster (13 min and 6.2 h, respectively) than previously published results for the intact MAb (mean t1/2β of 46 h). This represents a theoretical improvement in pharmacokinetics with respect to the parental murine MAb and points to the potential for utilizing this fragment in redirecting therapeutic agents, such as radioisotopes, to different human carcinomas overexpressing HER-2. Received: 10 August 2000 / Accepted: 19 October 2000     

Spermatogenesis in azoospermic,formerly cryptorchid men. Use of needle aspiration techniques

OBJECTIVE: To assess the use of testicular needle aspiration techniques to evaluate fertility potential in azoospermic, formerly cryptorchid men. STUDY DESIGN: Fifteen consecutive adult azoospermic, formerly cryptorchid patients (eight unilateral and seven bilateral) were examined by needle aspiration techniques, fine (FNA) and large needle (LNAB) testicular aspiration biopsy, for cytologic and histologic analysis. Five of the 15 subsequently underwent surgical biopsy for attempted assisted fertilization. RESULTS: Spermatozoa or spermatids were detected by FNA cytology or LNAB histology in one or both testicles in 87.5% of the unilateral and 28.6% of the bilaterally affected, formerly cryptorchid patients (P = .041, Fisher's exact test). The addition of LNAB to FNA identified spermatids in one patient with unilateral cryptorchidism and only Sertoli cells on FNA cytology. Furthermore, LNAB differentiated testicles with the cytologic finding of only Sertoli cells into those with or without diffuse fibrosis. In the five patients in whom assisted fertilization was attempted, the needle aspiration techniques predicted the presence or absence of spermatozoa in the subsequent surgical biopsy. CONCLUSION: The two needle aspiration techniques can be used to assess the fertility potential of azoospermic, formerly cryptorchid men and to select patients for assisted fertilization.     

Regulated gene expression of hyaluronan synthases during Xenopus laevis development

Here reported is the developmental gene expression pattern of the three known vertebrate hyaluronan synthases (XHas1, XHas2 and XHas3) and a comparative analysis of their mRNAs spatio-temporal distribution during Xenopus laevis development. We found that while XHas2 shows a steady-state expression from gastrula to late tailbud stage, XHas1 is mainly present in the early phases of development while XHas3 is predominantly transcribed in tailbud embryos. XHas1, XHas2 and XHas3 show distinct tissue expression patterns. In particular, XHas1 is localized in ectodermal derivatives and in cranial neural crest cells, whereas XHas2 is mainly found in mesoderm-derived structures and in trunk neural crest cells. Moreover, the expression pattern of XHas2 overlaps that of MyoD in cells committed to a muscle fate. Unlike the other hyaluronan synthases, XHas3 mRNA distribution is very restricted. In particular, XHas3 is expressed in the otic vesicles and closely follows the inner ear development. In conclusion, XHas1, XHas2 and XHas3 mRNAs have distinct and never overlapping spatial expression domains, which would suggest that these three enzymes may play different roles during embryogenesis.     

Functional and crystallographic characterization of Salmonella typhimurium Cu,Zn superoxide dismutase coded by the sodCI virulence gene

The functional and three-dimensional structural features of Cu,Zn superoxide dismutase coded by the Salmonella typhimurium sodCI gene, have been characterized. Measurements of the catalytic rate indicate that this enzyme is the most efficient superoxide dismutase analyzed so far, a feature that may be related to the exclusive association of the sodCI gene with the most pathogenic Salmonella serotypes. The enzyme active-site copper ion is highly accessible to external probes, as indicated by quenching of the water proton relaxation rate upon addition of iodide. The shape of the electron paramagnetic resonance spectrum is dependent on the frozen or liquid state of the enzyme solution, suggesting relative flexibility of the copper ion environment. The crystal structure (R-factor 22.6%, at 2.3 A resolution) indicates that the dimeric enzyme adopts the quaternary assembly typical of prokaryotic Cu,Zn superoxide dismutases. However, when compared to the structures of the homologous enzymes from Photobacterium leiognathi and Actinobacillus pleuropneumoniae, the subunit interface of Salmonella Cu,Zn superoxide dismutase shows substitution of 11 out of 19 interface residues. As a consequence, the network of structural water molecules that fill the dimer interface cavity is structured differently from the other dimeric bacterial enzymes. The crystallographic and functional characterization of this Salmonella Cu,Zn superoxide dismutase indicates that structural variability and catalytic efficiency are higher in prokaryotic than in the eukaryotic homologous enzymes.