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A critical feature of effective marine reserves is to be large enough to encompass home ranges of target species, thereby allowing a significant portion of the population to persist without the threat of exploitation. In this study, patterns of movement and home range for Lethrinus harak and Lethrinus obsoletus were quantified using an array of 33 acoustic receivers that covered approximately three quarters of Piti Marine Reserve in the Pacific island of Guam. This array was designed to ensure extensive overlap of receiver ranges throughout the study area. Eighteen individuals (12 L. harak and 6 L. obsoletus) were surgically implanted with ultrasonic transmitters and passively tracked for 4 months. Both species displayed high site fidelity and had relatively small home ranges. The home ranges of L. harak expanded with increasing body size. Feeding of fish by humans, which was common but restricted to a small area within the study site, had little effect on the distribution of the resident populations. L. harak made nightly spawning migrations within the reserve between full moon and last quarter moon of each lunar cycle, coinciding with a strong ebbing tide. Results indicate that even small reserves can include many individual home ranges of these emperorfishes and can protect spawning sites for L. harak. These species are heavily targeted in Guam, and there are major demographic differences between fished and protected sites. This study shows the potential for protected areas to sustain reproductive viability in exploited populations.

Marine reserves are the primary management tool for Guam’s reef fish fishery. While a build-up of fish biomass has occurred inside reserve boundaries, it is unknown whether reserve size matches the scale of movement of target species. Using passive acoustic telemetry, we quantified movement patterns and home range size of two heavily exploited unicornfish Naso unicornis and Naso lituratus. Fifteen fish (N. unicornis: n = 7; N. lituratus: n = 4 male, n = 4 female) were fitted with internal acoustic tags and tracked continuously over four months within a remote acoustic receiver array located in a decade-old marine reserve. This approach provided robust estimates of unicornfish movement patterns and home range size. The mean home range of 3.2 ha for N. unicornis was almost ten times larger than that previously recorded from a three-week tracking study of the species in Hawaii. While N. lituratus were smaller in body size, their mean home range (6.8 ha) was over twice that of N. unicornis. Both species displayed strong site fidelity, particularly during nocturnal and crepuscular periods. Although there was some overlap, individual movement patterns and home range size were highly variable within species and between sexes. N. unicornis home range increased with body size, and only the three largest fish home ranges extended into the deeper outer reef slope beyond the shallow reef flat. Both Naso species favoured habitat dominated by corals. Some individuals made predictable daily crepuscular migrations between different locations or habitat types. There was no evidence of significant spillover from the marine reserve into adjacent fished areas. Strong site fidelity coupled with negligible spillover suggests that small-scale reserves, with natural habitat boundaries to emigration, are effective in protecting localized unicornfish populations.

Hepadnaviruses have a complex replication cycle which includes reverse transcription of the pregenomic RNA. The initial step in this process in hepatitis B virus (HBV) requires the viral polymerase to engage a highly stable region of secondary structure within the pregenomic RNA termed the epsilon stem-loop. While reverse transcriptases belonging to the retrovirus family use a specific cellular tRNA as primer, HBV polymerase utilizes a tyrosine residue located within its own N terminus. Therefore, the first deoxyribonucleotide is covalently coupled to HBV polymerase prior to extension of the DNA strand by conventional reverse transcription. We have expressed HBV polymerase in a baculovirus and following purification have found it to be active with respect to protein-priming and reverse transcription of copurified RNA. Importantly, we found both of these processes to be critically dependent on the presence of the epsilon stem-loop. The metal ion preferences of HBV polymerase were also investigated for both the protein-priming and reverse transcription activities of this enzyme. Reverse transcription was dependent on magnesium, with an optimal concentration of 5 mM. However, protein-priming was strongly favoured by manganese ions and was optimal at a concentration of 1 mM. Thus, using manganese as sole source of metal ions our activity assay is restricted to the protein-priming event and will allow the search for novel antivirals specifically blocking this unique mechanism.

The recent approval by the regulatory authorities in the United States of several HIV proteinase inhibitors as therapeutics for the treatment of AIDS confirms that virus proteinases are valid molecular targets in the search for new antiviral drugs. This review summarizes the available approaches that can be taken to discover virus proteinase inhibitors and reviews the current status of our knowledge with respect to virus proteinases in viruses of clinical significance other than HIV. The major focus is on proteinases identified in the viruses that cause the common cold, hepatitis C virus and the herpesviruses.

After the U53 gene encoding the proteinase from human herpesvirus 6 (HHV-6) was sequenced, it was expressed in Escherichia coli, and the activity of the purified, recombinant HHV-6 proteinase was characterized quantitatively by using synthetic peptide substrates mimicking the release and maturation cleavage sites in the polyprotein precursors of HHV-6, human cytomegalovirus (CMV), murine CMV, and Epstein-Barr virus. Despite sharing 40% identity with other betaherpesvirus proteinases such as human CMV proteinase, the one-chain HHV-6 enzyme was distinguished from these two-chain proteinases by the absence of an internal autocatalytic cleavage site.

Hydroponic isotope labeling of entire plants (HILEP) combines hydroponic plant cultivation and metabolic labeling with stable isotopes using (15)N-containing inorganic salts to label whole and mature plants. Employing (15)N salts as the sole nitrogen source for HILEP leads to the production of healthy-looking plants which contain (15)N proteins labeled to nearly 100%. Therefore, HILEP is suitable for quantitative plant proteomic analysis, where plants are grown in either (14)N- or (15)N-hydroponic media and pooled when the biological samples are collected for relative proteome quantitation. The pooled (14)N-/(15)N-protein extracts can be fractionated in any suitable way and digested with a protease for shotgun proteomics, using typically reverse phase liquid chromatography nanoelectrospray ionization tandem mass spectrometry (RPLC-nESI-MS/MS). Best results were obtained with a hybrid ion trap/FT-MS mass spectrometer, combining high mass accuracy and sensitivity for the MS data acquisition with speed and high-throughput MS/MS data acquisition, increasing the number of proteins identified and quantified and improving protein quantitation. Peak processing and picking from raw MS data files, protein identification, and quantitation were performed in a highly automated way using integrated MS data analysis software with minimum manual intervention, thus easing the analytical workflow. In this methodology paper, we describe how to grow Arabidopsis plants hydroponically for isotope labeling using (15)N salts and how to quantitate the resulting proteomes using a convenient workflow that does not require extensive bioinformatics skills.

Genes were constructed to encode single-chain tethered human immunodeficiency virus HIV-1/HIV-1 and HIV-2/HIV-2 homodimeric proteinases and two HIV-1/HIV-2 heterodimers which differed in the nature of the interface strands. All four constructs under the control of a heat-inducible promoter were expressed in E. coli and the resultant proteinases were purified therefrom. Kinetic parameters (Km, kcat and kcat/Km) were derived for the interaction of the tethered homo and heterodimeric proteinases with two distinct substrates at a variety of pH values. All four enzymes were comparably active toward one substrate. With the second substrate at pH 4.7, the kcat/Km value was best for the HIV-1/1 tethered homodimer, 15-fold lower for the two heterodimeric proteinases, and was reduced by an additional 6-fold for the HIV-2/2 homodimer. From the Ki values determined for the interactions of the four tethered dimer proteinases with a systematic series of synthetic inhibitors, a parallel trend was observed. Whereas several inhibitors were equipotent against all four enzymes, two were discriminatory in that they inhibited strongly the HIV-1/1 homodimer and the two heterodimeric proteinases but had little effect on the HIV -2/2 tethered homodimer (or its untethered wild-type counterpart from HIV-2). The significance of these findings for active site interaction with HIV-proteinases is considered.

Activation of cGMP phosphodiesterase (PDE) by the rod G-protein transducin is a key event in visual signal transduction in vertebrate photoreceptor cells. Interaction between the GTP-bound form of the alpha-subunit of transducin (alpha t*) and the PDE inhibitory gamma-subunit (P gamma) is a major component of PDE activation. The central polycationic region of P gamma, P gamma-24-45, has been implicated as one of the sites involved in alpha t*.P gamma interaction. Here we determine the site on alpha t* that interacts with P gamma-24-45 using a photo-cross-linking approach. The synthetic peptides Cys(ACM)Tyr-P gamma-24-45-Cys (where ACM indicates acetamidomethyl group) and Cys-P gamma-24-45 were labeled with 4-(N-maleimido)benzophenone at the COOH and NH2 termini, respectively, and then cross-linked to alpha t. When the photoprobe was attached to the COOH terminus of the peptide, a specific high yield cross-linked product (80%) was formed between the peptide and alpha t GTP gamma S (guanosine 5'-O-(thiotriphosphate)). A lower yield of cross-linking (35%) was seen between the peptide and alpha t GDP. The site of cross-linking between Cys(ACM)Tyr-P gamma-24-45-Cys and alpha t GTP gamma S was localized to within alpha t-306-310 using a variety of chemical and proteolytic cleavages of the cross-linked product, analysis of the fragments with SDS-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry.

The human immunodeficiency virus (HIV) proteinase inhibitor Ro 31-8959 prevents the maturation of virus in phorbol 12-myristate 13-acetate (PMA)-stimulated U1 cells, a chronically infected promonocytic cell line. Inhibition of both the morphological maturation of virions and the enzymic processing of gag polyprotein (p56) to produce capsid protein p24 was demonstrated at nanomolar concentrations of the compound. Furthermore, prolonged inhibition of the processing of p56 antigen was confirmed in pulse-chase experiments. The conclusion is that Ro 31-8959 can inhibit production of mature virions in a promonocyte cell line which is infected chronically/latently with HIV.

We investigate possibilities of inducing temporal structures without fading memory in recurrent networks of spiking neurons strictly operating in the pulse-coding regime. We extend the existing gradient-based algorithm for training feedforward spiking neuron networks, SpikeProp (Bohte, Kok,La Poutré, 2002), to recurrent network topologies, so that temporal dependencies in the input stream are taken into account. It is shown that temporal structures with unbounded input memory specified by simple Moore machines (MM) can be induced by recurrent spiking neuron networks (RSNN). The networks are able to discover pulse-coded representations of abstract information processing states coding potentially unbounded histories of processed inputs. We show that it is often possible to extract from trained RSNN the target MM by grouping together similar spike trains appearing in the recurrent layer. Even when the target MM was not perfectly induced in a RSNN, the extraction procedure was able to reveal weaknesses of the induced mechanism and the extent to which the target machine had been learned.

The main aim of this study was to assess the level of agreement between the Eating Disorders Examination (EDE) and its self-report version (EDE-Q) on key items in a clinic sample of patients with bulimia nervosa. A second objective was to assess the concordance between self-reported and objective body weight in the sample.Sixty females who met DSM-IV criteria for bulimia nervosa (purging type) participated. Fifty-seven of them completed both the EDE and the EDE-Q. Self-reported weight was obtained during a telephone screening interview. Objective weight was subsequently measured at an assessment about a week later.The EDE generated higher scores than the EDE-Q for the frequency of objective binge and vomiting episodes. The two methods produced equivalent results for subjective binge episodes, laxative and diuretic misuse, and concerns about shape and weight. The self-report method underestimated body weight.These findings suggest that some core features of eating disorders are more accurately assessed using the EDE interview.

We propose that the N-formyl-Met-Leu-Phe binding site in the human neutrophil formyl peptide receptor (FPR) lies in the predicted transmembrane region. We examined the expression, binding, and G protein coupling of 28 mutated forms of FPR in stably transfected Chinese hamster ovary cells. The amino acids we mutated are: 1) predicted to be oriented toward the interhelical space; 2) analogous to those required for ligand binding in various other G protein-coupled receptors; 3) divergent from lipoxin A4 receptor, a low affinity receptor for formylated peptides; and 4) either highly conserved or divergent in other G protein-coupled receptors. Some mutations resulted in intracellular retention, suggesting that the receptors were misfolded. Most mutated receptors that were transported to the plasmalemma bound f-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with affinities similar to the wild-type receptor (Kd = 6 nM). However, mutations L78A (helix II), D106N, L109A (helix III), T157A (helix IV), R201A, I204Y, and R205A (helix V), W254A and Y257A (helix VI), and F291A (helix VII) resulted in reduced affinities (Kd = 30-128 nM). Of these mutations, D106N, R201A, and R205A also appeared to affect G protein coupling, suggesting that these residues may also be involved in signal transduction and/or are essential for proper folding of the molecule. Some of the FPR residues that appeared to be involved in binding of formylated peptides were located at sites analogous to those identified in ligand binding to certain other G protein-coupled receptors. It is thus possible that several G protein-coupled receptors have a common placement of ligand-binding amino acids.

Varicella-zoster virus (VZV) genes 33 and 33.5 are predicted to encode the VZV proteinase and its substrate (the assembly protein) respectively. These genes were expressed in insect cells using recombinant baculovirus and it was confirmed that gene 33 encodes a proteinase capable of autoproteolytic processing at two positions. When VZV gene 33.5 was co-expressed with the VZV proteinase, processing of the VZV33.5 gene product was observed. A polyclonal antiserum to the VZV assembly protein domain highlighted a set of proteins in VZV infected HEL cells identical to those identified in insect cells expressing VZV genes 33 and 33.5. To facilitate further characterization of the VZV proteinase the enzyme was purified by affinity chromatography from an E. coli expression system and in vitro activity was observed.

The herpes simplex virus VP16 protein functions as a potent transcriptional activator and targets DNA sites with the consensus TAATGARAT present in all the viral immediate-early gene promoters. To do so, VP16 directs assembly of a multiprotein complex involving two cellular proteins, host cell factor (HCF) and the Oct-1 DNA-binding transcription factor. To investigate the importance of specific protein-protein interactions to formation of this VP16-induced complex (VIC), we used oligopeptides to prevent VIC assembly. Linear and cyclic peptides corresponding to a region of VP16 previously implicated in complex formation were potent inhibitors of VIC assembly. To further characterize the protein interactions involved, we cloned a human cDNA encoding the minimal VP16 interaction domain of HCF, containing amino acids 1 to 380 [HCF (1-380)]. The REHAYS-based peptides active in preventing VIC assembly were found to specifically block binding of VP16 to HCF (1-380), without affecting VP16-Oct-1 binding. The inhibitory activity of these VP16 peptides was strictly sequence specific for the EHAY residues. Site-directed mutagenesis of the HCF (1-380) domain revealed residues E102 and K105 to be critical determinants in support of VIC formation. Alteration of a single residue in HCF, K105, was shown to virtually abolish complex assembly. Interestingly however, none of the HCF mutants that were impaired in their ability to support complex formation exhibited defects in direct VP16 binding, supporting loss of function at a higher order in complex assembly.

Genes were constructed to encode single-chain tethered human immunodeficiency virus HIV-1/HIV-1 and HIV-2/HIV-2 homodimeric proteinases and two HIV-1/HIV-2 heterodimers which differed in the nature of the interface strands. All four constructs under the control of a heat-inducible promoter were expressed in E. coli and the resultant proteinases were purified therefrom. Kinetic parameters (Km, kcat and kcat/Km) were derived for the interaction of the tethered homo and heterodimeric proteinases with two distinct substrates at a variety of pH values. All four enzymes were comparably active toward one substrate. With the second substrate at pH 4.7, the kcat/Km value was best for the HIV-1/1 tethered homodimer, 15-fold lower for the two heterodimeric proteinases, and was reduced by an additional 6-fold for the HIV-2/2 homodimer. From the Ki values determined for the interactions of the four tethered dimer proteinases with a systematic series of synthetic inhibitors, a parallel trend was observed. Whereas several inhibitors were equipotent against all four enzymes, two were discriminatory in that they inhibited strongly the HIV-1/1 homodimer and the two heterodimeric proteinases but had little effect on the HIV -2/2 tethered homodimer (or its untethered wild-type counterpart from HIV-2). The significance of these findings for active site interaction with HIV-proteinases is considered.

Activation of cGMP phosphodiesterase (PDE) by the rod G-protein transducin is a key event in visual signal transduction in vertebrate photoreceptor cells. Interaction between the GTP-bound form of the alpha-subunit of transducin (alpha t*) and the PDE inhibitory gamma-subunit (P gamma) is a major component of PDE activation. The central polycationic region of P gamma, P gamma-24-45, has been implicated as one of the sites involved in alpha t*.P gamma interaction. Here we determine the site on alpha t* that interacts with P gamma-24-45 using a photo-cross-linking approach. The synthetic peptides Cys(ACM)Tyr-P gamma-24-45-Cys (where ACM indicates acetamidomethyl group) and Cys-P gamma-24-45 were labeled with 4-(N-maleimido)benzophenone at the COOH and NH2 termini, respectively, and then cross-linked to alpha t. When the photoprobe was attached to the COOH terminus of the peptide, a specific high yield cross-linked product (80%) was formed between the peptide and alpha t GTP gamma S (guanosine 5'-O-(thiotriphosphate)). A lower yield of cross-linking (35%) was seen between the peptide and alpha t GDP. The site of cross-linking between Cys(ACM)Tyr-P gamma-24-45-Cys and alpha t GTP gamma S was localized to within alpha t-306-310 using a variety of chemical and proteolytic cleavages of the cross-linked product, analysis of the fragments with SDS-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry.

In photoreceptor cells of vertebrates light activates a series of protein-protein interactions resulting in activation of a cGMP-phosphodiesterase (PDE). Interaction between the GTP-bound form of rod G-protein alpha-subunit (alpha t) and PDE inhibitory gamma-subunit (P gamma) is a key event for effector enzyme activation. This interaction has been studied using P gamma labeled with the fluorescent probe, lucifer yellow vinyl sulfone, at Cys-68 (P gamma LY) and sites of interaction on alpha t and P gamma have been investigated. Addition of alpha tGTP gamma S to P gamma LY produced a 3.2-fold increase in the fluorescence of P gamma LY. The Kd for alpha tGTP gamma S.P gamma LY interaction was 36 nM. Addition of 1 microM alpha tGDP had no effect, but in the presence of A1F4-, alpha tGDP increased P gamma LY fluorescence by 85%. When P gamma LY was reconstituted with P alpha beta to form fluorescent holo-PDE, alpha tGTP gamma S increased the fluorescence of holo-PDE with a K0.5 = 0.7 microM. Also, alpha tGTP gamma S stimulated the activity of this PDE over an identical range of concentrations with a similar K0.5 (0.6 microM). alpha tGTP gamma S enhanced the fluorescence of a COOH-terminal P gamma fragment, P gamma LY-46-87, as well (Kd = 1.5 microM). We demonstrate that an alpha t peptide, alpha t-293-314, which activated PDE (Rarick, H. M., Artemyev, N. O., and Hamm, H. E. (1992) Science 256, 1031-1033), mediates PDE activation by interacting with the P gamma-46-87 region. Peptide alpha t-293-314 bound to P gamma LY (K0.5 = 1.2 microM) as well as to the carboxyl-terminal P gamma fragment, P gamma LY-46-87 (K0.5 = 1.7 microM) as measured by fluorescence increase, while other alpha t peptides had no effect. A peptide from the P gamma central region, P gamma-24-46, blocked the interaction between alpha tGTP gamma S and P gamma LY. The Kd for alpha tGTP gamma S.P gamma-24-46 interaction was 0.7 microM. On the other hand, P gamma-24-46 had no effect on alpha t-293-314 interaction with P gamma LY. Our data suggest that there are at least two distinct sites of interaction between alpha tGTP gamma S and P gamma. The interaction between alpha t-293-314 and P gamma-46-87 is important for PDE activation.(ABSTRACT TRUNCATED AT 400 WORDS)

The binding of dihydropyridine (PN200-110) to skeletal muscle microsomes (which were 84% sealed inside-out vesicles) was not influenced by the addition of calcium or magnesium nor by addition of their chelators (EDTA or EGTA) unless the vesicles were pretreated with the calcium-magnesium ionophore A23187 and EDTA to remove entrapped cations. Separation of inside-out vesicles from right-side-out vesicles by wheat germ agglutinin chromatography revealed that only the right-side-out vesicles exhibited a calcium-, magnesium-, and chelator-dependent binding of PN200-110. Dihydropyridine binding to cardiac sarcolemma membranes (which were 46% inside-out) and to solubilized skeletal muscle membranes was inhibited by EDTA and could be fully restored by 10 microM calcium or 1 mM magnesium. Calcium increased PN200-110 binding to partially purified rabbit skeletal muscle calcium channels from 3.9 pmol/mg protein to 25.5 pmol/mg protein with a pK0.5 = 6.57 +/- 0.059 and a Hill coefficient of 0.56 +/- 0.04. Magnesium increased binding from 0.7 pmol/mg protein to 16.8 pmol/mg protein with a pK0.5 = 3.88 +/- 0.085 and a Hill coefficient of 0.68 +/- 0.074. These studies suggest that calcium binding to high affinity sites or magnesium binding to low affinity sites on the extracellular side of skeletal muscle T-tubule calcium channels regulates dihydropyridine binding. Further, similar calcium and magnesium binding sites exist on the cardiac calcium channel and serve to allosterically regulate dihydropyridine binding.

Spinach calmodulin (CaM) has been labeled at cysteine-26 with the sulfhydryl-selective probe 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) to produce MIANS-CaM. The interaction of MIANS-CaM with CaM binding proteins was studied by fluorescence enhancement accompanying the protein-protein interactions. MIANS-CaM bound to smooth muscle myosin light-chain kinase with a Kd of 9 nM, causing a 4.6-fold fluorescence enhancement. Caldesmon bound with a Kd of 250 nM, causing a 2-fold fluorescence enhancement. Calcineurin (CaN) bound to MIANS-CaM with a Kd less than 5 nM, causing an 80% increase in fluorescence. On the other hand, binding of the CaM antagonist drugs prenylamine and calmidazolium or the potent peptide antagonist melittin did not alter MIANS fluorescence. MIANS-CaM activated brain cGMP phosphodiesterase and CaN as effectively as unlabeled CaM. Spinach CaM was also labeled with three other sulfhydryl reagents, 6-acryloyl-2-(dimethylamino)naphthalene, (2,5-dimethoxy-4-stilbenyl)maleimide, and rhodamine X maleimide. CaN bound to the highly fluorescent rhodamine X maleimidyl-CaM with a Kd of 1.4 nM, causing a 25% increase in polarization. Both MIANS-CaM and rhodamine X-CaM were used to monitor the Ca2+ dependence of the interaction between CaM and CaN. Half-maximal binding occurred at pCa 6.7-6.8 in the absence of Mg2+, or at pCa 6.3 in the presence of 3 mM Mg2+. In both cases, the dependence of the interaction was cooperative with respect to Ca2+ (Hill coefficients of 1.7-2.0). Use of these fluorescent CaMs should allow accurate monitoring of CaM interactions with its target proteins and perhaps their localization within the cell.

Two flights from Alice Springs, Australia, were achieved in November 1977 and November 1978 with a plastic scintillator γ-burst detector, effective area 6.3 m2, thickness 5 cm, energy response in the range 50 keV to 2 MeV. In 33 hr of good, high altitude data, two bursts were detected, yielding a rate corrected to an isotropic flux of\(7.03 \times 10^3 {\text{y}}^{ - 1} \left\{ {\begin{array}{*{20}c} { + 10 \times 10^3 } \\ { - 6 \times 10^3 } \\ \end{array} } \right\}\) at a size of 8.5×10−9 erg cm−2. One event, seen at 22.14 on 15 Nov 1978, was confirmed by spacecraft measurements. The second, too small to be detected by spacecraft, arrived from 0 hr RA, −13.2° Decl. ±12° and possibly comes from a confirmed γ-burst source location. A galactic origin with a source distribution originating from a relatively thick disk, is favoured by these results.

Studies of the propensity of aviation turbine fuels to lacquer engine oil-coolers that were described in an earlier paper have been extended to cover a wider range of fuels. Fuel performance was found to vary widely; some fuels were liable to lacquer oil-coolers to the extent of producing significant losses in efficiency at the most severe operating conditions currently encountered. Oxidation studies conducted in parallel with the rig investigations indicate that a fuel’s performance is strongly dependent on its tendency to initiate radical oxidation reactions. The relatively high initiation rate of less stable fuels is believed to be due in part to their trace content of metals that catalyze oxidation reactions. Accordingly, an approved metal deactivating additive has been examined as a means of improving the performance of such fuels.

Previously we have shown that the fluorescence of the dihydropyridine calcium antagonist felodipine provides an accurate means of monitoring the formation of an allosterically potentiated conformer of calmodulin (Mills, J. S., and Johnson, J. D., (1985) Biochemistry 24, 4897-4903). Characteristic of this conformer is the abolition of cooperativity among the two felodipine-binding sites on calmodulin and a 20-fold increase in the apparent affinity of calmodulin for felodipine. In the present study, we find that the metal cations La3+, Tb3+, Pb2+, and Cd2+ are all capable of abolishing the cooperativity (Hill coefficient = 2.0) among the two felodipine-binding sites on calmodulin and can increase the apparent affinity of calmodulin for felodipine by approximately 20-fold. These effects are seen either in the presence or absence of calcium and are half-maximal at 8, 12, 22, and 1000 microM, respectively. Zinc and H+ are capable of producing similar potentiations of felodipine binding (half-maximal at 570 microM, and pH 5.8), but only in the presence of calcium. In each case, the calcium-binding sites of calmodulin must be occupied (by calcium, La3+, Tb3+, Pb2+, or by Cd2+) before these metals can bind to sites which are distinct from the calcium-binding sites to produce the active conformer of calmodulin which exhibits enhanced affinity for felodipine. Mercury and copper can compete with these potentiating metal cations on calmodulin and produce an inactivation of this active calmodulin conformer. These studies suggest that some metals including La3+, Tb3+, Pb2+, Cd2+, Zn2+ and protons are capable of binding to a calcium-calmodulin complex and forming an allosterically active species of calmodulin which cannot be maintained by physiological concentrations of calcium ions alone. Mercury and copper, on the other hand, are capable of inactivating this active calmodulin conformer independent of the presence of calcium on calmodulin. These findings are examined in terms of the mechanism of action of calmodulin and its possible role in heavy metal toxicity.

The effects of reactor exposures in the range 1016?1018 fast neutrons per cm2 (square centimeter) on the magnetic properties of 16 polycrystalline ferrites and garnets are presented. Measured properties included coercive force, remanence, saturation induction, complex permeability, and FMR (ferromagnetic resonance) linewidth. It was found that the hysteresis properties and FMR linewidth were slightly affected by exposures of 1018 neutrons per cm2. Complex permeability was affected by exposures as small as 1016 neutrons per cm2. All of the observed changes were small.

WE report on some bursts of hard X rays seen in March 1976 by the Ariel-V scintillation telescope (ST). The timescale of a few seconds, the correlation with low energy events seen by a proportional counter on the same satellite (ref. 1 and S. J. Bell-Burnell, private communication) and a photon energy > 50 keV, are factors which suggest a connection in origin between the gamma bursts of Klebesadel et al.2 and the X-ray events noticed by Grindlay et al.3. A delayed arrival at high energies seems to be an important feature of our observations.

To obtain more data at the low flux end of the size spectrum and in particular to both lower the size limit of burst detection and look for a possible excess flux from the galactic centre region, large-area detector flights were undertaken from Australia. The apparatus consisted of plastic scintillators, 5 cm thick and 6.3 m2 in effective area, arranged in six trays, two of which were horizontal and four of which were inclined at 30? to the horizontal so as to make up the four sloping sides of a box. Laboratory calibration gave a mean efficiency for the detection of 50 keV to 2 MeV photons of 0.404 when translated to 4 g cm-2 atmospheric depth and for all bursts arriving up to a cut-off zenith angle of 60?. Thirty-three hours of good data were obtained in total during flights in November 1977 and November 1978. Gamma-burst searches were made by demanding three successive 2o increases in at least three of the six trays with a sampling interval of 1/16s. Two bursts were detected, one at 1978, 15 November, 22h14, in coincidence with Venera spacecraft observations of an event, and the second at 1978, 16 November, 13h40 at a flux level of 9.2 x 10-8 erg cm-2. Directional determination for this second burst with the inclined tray rates gave I- 82.5?, b = - 71.4?, with an error circle of 12'. A Venera event occurred on 19 November 1978, 24' away from this position. Correction of the flight results to an isotropic flux, missed short bursts, the flat detector response and missing energy being taken into account, on the assumption of the Apollo 16 (Mezger et al. I974) spectrum, yielded a rate (7+10) x 103 year-1 at sizes greater than or equal to 8.47 x 10-9 erg cm-2. This point is plotted

The electron spin resonance spectra of solutions of nitric oxide-hemoglobin and nitric oxide-methemoglobin, and whole blood treated at room temperature with nitric oxide, all exhibit resonance with a line width of 83 gauss, a g-value of 2.03, and a spin intensity corresponding to one unpaired electron spin per heme. The minimum detectable concentration of these nitric oxide complexes in solution is 10(-5)M. Solutions were stable in a nitrogen atmosphere but when exposed to air in the absence of nitric oxide the spin intensity decreased with a half-life of about 5 hours. A preliminary examination of blood of rats exposed 1 and 9 days to 10 ppm of nitric oxide in air showed no electron spin resonance.

The propensity of aviation turbine fuels to produce deposits in the oil-cooler and filter sections of aircraft fuel systems has been examined using a rig that simulates the fuel system of an aircraft and which employs realistic flow rates. All the fuels examined were found to be thermally stable up to temperatures in excess of those currently attained in engine oil coolers. Comparison with results obtained with the JFTOT indicates that this is not suited for use as a research tool.Copyright © 1984 by ASME

Six laminate configurations were subjected to five different thermal exposures in the temperature range 78K to 603K (−320°F to 625°F), and then studied using microscopy and x-ray to determine the characteristics of microcracks formed during the thermal loadings. The laminates studied were: [03903]s, [02/902]s, [(0/90)3]s, [45/−45/0/90]s, [0/45/90/−45]s, and [0/60/0/−60]s. The material system investigated was found to be free of cracks after curing, but microcracks did develop in most laminates when cooled from 603K (625°F) by quenching in ice water or liquid nitrogen. Crack density was dependent on laminate configuration and rate of cooling. Microcracks present at free edges extended across the entire width of the specimens. The [45/−45/0/90]s laminate proved to be very resistant to microcracking for all thermal loadings. The thermal load required to initiate microcracking, determined using laminate analysis with stress and temperature dependent material properties, compared reasonably well with experimental results.

Previously we have shown that the fluorescence of the dihydropyridine calcium antagonist felodipine provides an accurate means of monitoring the formation of an allosterically potentiated conformer of calmodulin (Mills, J. S., and Johnson, J. D., (1985) Biochemistry 24, 4897-4903). Characteristic of this conformer is the abolition of cooperativity among the two felodipine-binding sites on calmodulin and a 20-fold increase in the apparent affinity of calmodulin for felodipine. In the present study, we find that the metal cations La3+, Tb3+, Pb2+, and Cd2+ are all capable of abolishing the cooperativity (Hill coefficient = 2.0) among the two felodipine-binding sites on calmodulin and can increase the apparent affinity of calmodulin for felodipine by approximately 20-fold. These effects are seen either in the presence or absence of calcium and are half-maximal at 8, 12, 22, and 1000 microM, respectively. Zinc and H+ are capable of producing similar potentiations of felodipine binding (half-maximal at 570 microM, and pH 5.8), but only in the presence of calcium. In each case, the calcium-binding sites of calmodulin must be occupied (by calcium, La3+, Tb3+, Pb2+, or by Cd2+) before these metals can bind to sites which are distinct from the calcium-binding sites to produce the active conformer of calmodulin which exhibits enhanced affinity for felodipine. Mercury and copper can compete with these potentiating metal cations on calmodulin and produce an inactivation of this active calmodulin conformer. These studies suggest that some metals including La3+, Tb3+, Pb2+, Cd2+, Zn2+ and protons are capable of binding to a calcium-calmodulin complex and forming an allosterically active species of calmodulin which cannot be maintained by physiological concentrations of calcium ions alone. Mercury and copper, on the other hand, are capable of inactivating this active calmodulin conformer independent of the presence of calcium on calmodulin. These findings are examined in terms of the mechanism of action of calmodulin and its possible role in heavy metal toxicity.

A full length cDNA clone encoding a mouse prostatic secretory glycoprotein (p12) whose synthesis is dependent upon testicular androgens has been cloned and characterized. The predicted amino acid sequence of p12 shares extensive homology with several members of the Kazal family of secretory protease inhibitors, in particular the pancreatic secretory trypsin inhibitors. In agreement with sequence data, prostatic secretory p12, purified from mouse ventral prostate secretion, exhibits anti-trypsin activity. Steady-state levels of protease inhibitor mRNA in ventral prostate are reduced from approximately 0.06% in normal mice to undetectable after androgen withdrawal but are inducible within 4 h by re-administration of testosterone. Androgen-dependent expression of the secretory protease inhibitor mRNA was also observed in coagulating gland and seminal vesicle. In seminal vesicle, a tissue of different embryonic origin to the prostate, the kinetics of secretory protease inhibitor mRNA loss after castration are not as rapid as in the ventral prostate and coagulating gland. Low-level androgen independent expression was also observed in the pancreas. There appears to be a single gene for this secretory protease inhibitor and yet expression is markedly stimulated by testosterone in the sex accessory tissues and unaffected by this hormone in the pancreas.

WE report on the combined results of three balloon-borne searches for small cosmic γ-ray bursts, carried out by the Imperial College Space Physics Groups. We suggest that our results are incompatible with the extragalactic source postulated for the large events previously seen by satellites. No decisive directional information has been obtained from the timing measurements, carried out by the Vela satellites on the large (10–100 photon cm−2s−1 (> 100 keV)) events, although a weak correlation with the local spiral arm exists1.

THE chemical composition of succinite (Baltic amber), its botanical origin, and methods of distinguishing it from other fossil resins, are long standing questions1, the third of which has been largely solved in recent years by infrared spectrometry2–4. In his survey, Langenheim5 emphasizes the botanical origin and the strong hold which Conwentz's postulated amber source Pinus succinifera has had over subsequent workers. This concept has also dominated ideas of the resin's composition; recent suggestions that this largely derives from dimerized abietic acid6,7 are not persuasive.