An NMR study of the interaction between the human copper(I) chaperone and the second and fifth metal-binding domains of the Menkes protein

The interaction between the human copper(I) chaperone, HAH1, and one of its two physiological partners, the Menkes disease protein (ATP7A), was investigated in solution using heteronuclear NMR. The study was carried out through titrations involving HAH1 and either the second or the fifth soluble domains of ATP7A (MNK2 and MNK5, respectively), in the presence of copper(I). The copper-transfer properties of MNK2 and MNK5 are similar, and differ significantly from those previously observed for the yeast homologous system. In particular, no stable adduct is formed between either of the MNK domains and HAH1. The copper(I) transfer reaction is slow on the time scale of the NMR chemical shift, and the equilibrium is significantly shifted towards the formation of copper(I)-MNK2/MNK5. The solution structures of both apo- and copper(I)-MNK5, which were not available, are also reported. The results are discussed in comparison with the data available in the literature for the interaction between HAH1 and its partners from other spectroscopic techniques.     

Opposite effects of Ca(2+) and GTP binding on tissue transglutaminase tertiary structure

Tissue transglutaminase (tTG) belongs to a class of enzymes that catalyze a cross-linking reaction between proteins or peptides. The protein activity is known to be finely tuned by Ca(2+) and GTP binding. In this study we report the effects of these ligands on the enzyme structure, as revealed by circular dichroism, and steady-state and dynamic fluorescence measurements. We have found that calcium and GTP induced opposite conformational changes at the level of the protein tertiary structure. In particular the metal ions were responsible for a small widening of the protein molecule, as indicated by anisotropy decay measurements and by the binding of a hydrophobic probe such as 1-anilino-8-naphthalenesulfonic acid (ANS). Unlike Ca(2+), the nucleotide binding increased the protein dynamics, reducing its rotational correlation lifetime from 32 to 25 ns, preventing also the binding of ANS into the protein matrix. Unfolding of tTG by guanidinium hydrochloride yielded a three-state denaturation mechanism, involving an intermediate species with the characteristics of the so-called "molten globule" state. The effect of GTP binding (but not that of Ca(2+)) had an important consequence on the stability of tissue transglutaminase, increasing the free energy change from the native to the intermediate species by at least approximately 0.7 kcal/mol. Also a greater stability of tTG to high hydrostatic pressure was obtained in presence of GTP. These findings suggest that the molecular mechanism by which tTG activity is inhibited by GTP is essentially due to a protein conformational change which, decreasing the accessibility of the protein matrix to the solvent, renders more difficult the exposure of the active site.     

FISH detection of ribosomal cistrons and assortment-distortion for X and B chromosomes in Dichroplus pratensis (Acrididae)

Assortment-distortion with respect to the X and NOR activity of a rare mitotically stable B chromosome (B(N)), was examined in 16 males of Dichroplus pratensis (Acrididae: Melanoplinae) from Argentine populations. In 1B individuals, the X and B associate preferentially during prophase I reaching a maximum level of association at zygotene. Frequency of X/B association remains relatively high up to diplotene-diakinesis and decreases steeply towards metaphase I. The percent X/B association at each stage is positively influenced by association at the previous stage, and interindividual variability in X/B association decreases as the frequency of association increases. Both chromosomes tended to preferentially orientate toward the same pole at MI (mean ratio of 16 individuals, 1.50:1) which determined an excess of XB and 00 second spermatocytes over X0 and 0B ones (1.39:1). No significant differences occurred between the MI, AI and MII assortment ratios. Fluorescent in situ hybridisation (FISH) confirmed that the B chromosome carries ribosomal genes and helped to establish that, during spermiogenesis, both the B and the normal NOR-bearing chromosome (S8) are clustered near the centriole adjunct region of spermatids. However, FISH failed to reveal the existence of inactive ribosomal cistrons in the X chromosome, as previously suggested, thus providing no support to a simple origin of the B from the X.     

Role of the tertiary and quaternary structures in the stability of dimeric copper, zinc superoxide dismutases

The equilibrium unfolding process of human Cu,Zn superoxide dismutase has been quantitatively monitored through circular dichroism and fluorescence spectroscopy as a function of increasing guanidinium hydrochloride concentration. The process occurs through the formation of a monomeric intermediate species following a three-state transition equilibrium. Comparison with the stability of the prokaryotic Cu,Zn SOD from P. leiognathi shows that the eukaryotic enzyme is more stable than the prokaryotic enzyme by approximately 3 kcal/mol. This difference is due to the monomer-to-unfolded equilibrium, while the dimer-to-monomer equilibrium is comparable for the two enzymes despite their different intersubunit interactions. These results are confirmed by the unfolding of the copper-depleted derivatives. The Cu,Zn superoxide dismutase represents a good example of how evolution has found two independent quaternary assemblies maintaining the same dimer stability.     

The cryoprotectant used, its concentration, and the equilibration time are critical for the successful cryopreservation of rabbit sperm: Dimethylacetamide versus dimethylsulfoxide

This study was designed to identify a suitable freezing protocol for rabbit semen by comparing the effects of different concentrations and equilibration times of dimethylacetamide (DMA) and dimethylsulfoxide (DMSO) on the postthaw quality of the semen. After establishing the best protocols for each cryoprotectant, their efficacy was compared by examining the in vivo fertilizing capacity of the semen samples. Pooled semen samples diluted in freezing medium containing 4%, 6%, or 8% DMA or DMSO (all combined with 1% sucrose as a nonpermeating cryoprotectant) were loaded in straws and equilibrated for 5, 15, or 45 min before freezing in liquid nitrogen vapor. The variables assessed after thawing were sperm motility, viability, osmotic resistance, and acrosome and DNA integrity. Marked effects on these variables were shown by the cryoprotectant concentration and equilibration time, with best results obtained using DMA 6% or DMSO 8% and equilibration times of 45 min. These freezing protocols were selected to compare the two cryoprotectants in an insemination trial. Three groups of 114 rabbit does (28 nulliparous and 86 multiparous in each group) were inseminated with fresh semen or with semen frozen using the optimized DMA or DMSO protocols. Fertility rates and numbers of kids born were similar, respectively for the DMSO-frozen (79.8% and 7.7 ± 0.3 young per kindling) and fresh semen (81.6% and 8.6 ± 0.3) yet higher (P ≤ 0.05) than the rates returned using the DMA-frozen semen (47.4% and 6.7 ± 0.4). Moreover, the numbers of rabbits born alive when DMSO was used in the freezing protocol, despite being lower than those recorded using fresh semen, were higher than when DMA was used as the cryoprotectant (P < 0.05). The physiological status of the does (nulliparous or multiparous) had no influence on the fertility and prolificacy results. Our findings indicate that the cryosurvival of rabbit sperm frozen using DMSO or DMA as the cryoprotectant is highly influenced by the concentration of cryoprotectant used and the time the semen is exposed to the agent before freezing. According to our in vivo fertility and prolificacy data, DMSO emerged as more effective than DMA for the cryopreservation of rabbit sperm.     

Targeting of PBP1 by β-lactams Determines recA/SOS Response Activation in Heterogeneous MRSA Clinical Strains

The SOS response, a conserved regulatory network in bacteria that is induced in response to DNA damage, has been shown to be associated with the emergence of resistance to antibiotics. Previously, we demonstrated that heterogeneous (HeR) MRSA strains, when exposed to sub-inhibitory concentrations of oxacillin, were able to express a homogeneous high level of resistance (HoR). Moreover, we showed that oxacillin appeared to be the triggering factor of a β-lactam-mediated SOS response through lexA/recA regulators, responsible for an increased mutation rate and selection of a HoR derivative. In this work, we demonstrated, by selectively exposing to β-lactam and non-β-lactam cell wall inhibitors, that PBP1 plays a critical role in SOS-mediated recA activation and HeR-HoR selection. Functional analysis of PBP1 using an inducible PBP1-specific antisense construct showed that PBP1 depletion abolished both β-lactam-induced recA expression/activation and increased mutation rates during HeR/HoR selection. Furthermore, based on the observation that HeR/HoR selection is accompanied by compensatory increases in the expression of PBP1,-2, -2a, and -4, our study provides evidence that a combination of agents simultaneously targeting PBP1 and either PBP2 or PBP2a showed both in-vitro and in-vivo efficacy, thereby representing a therapeutic option for the treatment of highly resistant HoR-MRSA strains. The information gathered from these studies contributes to our understanding of β-lactam-mediated HeR/HoR selection and provides new insights, based on β-lactam synergistic combinations, that mitigate drug resistance for the treatment of MRSA infections.     

Antitumour efficacy of lymphokine-activated killer cells loaded with ricin against experimentally induced lung metastases

Summary Adoptive transfer of tumour-specific T lymphocytes loaded with ricin into tumour-bearing mice exerts a transient therapeutic effect against locally induced tumours [Cerundolo et al. (1987) Br J Cancer 55: 413]. As transferred cells preferentially locate in the lung, we studied the therapeutic effect of ricin-loaded, lymphokine-activated killer (LAK) cells on lung metastases induced by M4 or B16-F1 (F1) tumour cell injection. In vitro studies demonstrated that ricin-treated LAK cells were about 100-fold more efficient than untreated LAK cells in inhibiting growth of the ricin-sensitive M4 tumour cell line. This effect was most likely due to the released ricin, as treated and untreated LAK cells inhibited the relatively toxin-resistant F1 cell line to the same extent. Ricin treatment did not alter the tissue distribution of intravenously (i.v.) injected LAK cells, which selectively localized in the lung early after inoculation, whether or not metastases were present. Adoptive transfer experiments showed that ricintreated LAK cells were significantly more efficient than untreated LAK cells in inhibiting M4- but not F1-induced lung metastases. These results indicate that LAK cells are able to deliver a therapeutic concentration of antineoplastic compounds directly to the lung.Research activities were partially developed in relation to a contract with the National Program of Pharmacological Research (Rif. 078606), by the Italian Consortium for Antitumoral Vectors (C.I.V.A) for the Italia Ministry of University and Scientific and Technological Research     

Filamentous Proteins from Plant Sieve Tubes

PROTEIN filaments are characteristic structural components of the assimilatory conducting elements of angiosperm plants (“P protein” of Cronshaw and Esau¹). We have isolated filamentous structures from the phloem exudate of cut cucurbit stems². The presence of the filaments could be clearly demonstrated after negative staining with the electron microscope.     

Analysis of locomotor activity rhythms in Drosophila

The genetic, molecular and anatomical dissection of the circadian clock in Drosophila and other higher organisms relies on the quantification of rhythmic phenotypes. Here, we introduce the methods currently in use in our laboratories for the analysis of fly locomotor activity rhythms. This phenotype provides a relatively simple, automated, efficient, reliable and robust output for the circadian clock. Thus it is not surprising that it is the preferred readout for measuring rhythmicity under a variety of conditions for most fly clock laboratories. The procedure requires at least 10 days of data collection and several days for analysis. In this protocol we advise on fly maintenance and on experimental design when studying the genetics of behavioral traits. We describe the setup for studying locomotor activity rhythms in the fruit fly and we introduce the statistical methods in use in our laboratories for the analysis of periodic data.     

Nonopioid placebo analgesia is mediated by CB1 cannabinoid receptors

Placebo analgesia is mediated by both opioid and nonopioid mechanisms, but so far nothing is known about the nonopioid component. Here we show that the specific CB1 cannabinoid receptor antagonist 5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (rimonabant or SR141716) blocks nonopioid placebo analgesic responses but has no effect on opioid placebo responses. These findings suggest that the endocannabinoid system has a pivotal role in placebo analgesia in some circumstances when the opioid system is not involved.