The Dimorphos ejecta plume properties revealed by LICIACube.

The Double Asteroid Redirection Test (DART) had an impact with Dimorphos (a satellite of the asteroid Didymos) on 26 September 20221. Ground-based observations showed that the Didymos system brightened by a factor of 8.3 after the impact because of ejecta, returning to the pre-impact brightness 23.7 days afterwards2. Hubble Space Telescope observations made from 15 minutes after impact to 18.5 days after, with a spatial resolution of 2.1 kilometres per pixel, showed a complex evolution of the ejecta3, consistent with other asteroid impact events. The momentum enhancement factor, determined using the measured binary period change4, ranges between 2.2 and 4.9, depending on the assumptions about the mass and density of Dimorphos5. Here we report observations from the LUKE and LEIA instruments on the LICIACube cube satellite, which was deployed 15 days in advance of the impact of DART. Data were taken from 71 seconds before the impact until 320 seconds afterwards. The ejecta plume was a cone with an aperture angle of 140 ± 4 degrees. The inner region of the plume was blue, becoming redder with increasing distance from Dimorphos. The ejecta plume exhibited a complex and inhomogeneous structure, characterized by filaments, dust grains and single or clustered boulders. The ejecta velocities ranged from a few tens of metres per second to about 500 metres per second.

Measurement of Jupiter's asymmetric gravity field.

The gravity harmonics of a fluid, rotating planet can be decomposed into static components arising from solid-body rotation and dynamic components arising from flows. In the absence of internal dynamics, the gravity field is axially and hemispherically symmetric and is dominated by even zonal gravity harmonics J2n that are approximately proportional to qn, where q is the ratio between centrifugal acceleration and gravity at the planet's equator. Any asymmetry in the gravity field is attributed to differential rotation and deep atmospheric flows. The odd harmonics, J3, J5, J7, J9 and higher, are a measure of the depth of the winds in the different zones of the atmosphere. Here we report measurements of Jupiter's gravity harmonics (both even and odd) through precise Doppler tracking of the Juno spacecraft in its polar orbit around Jupiter. We find a north-south asymmetry, which is a signature of atmospheric and interior flows. Analysis of the harmonics, described in two accompanying papers, provides the vertical profile of the winds and precise constraints for the depth of Jupiter's dynamical atmosphere.

Glucose-6-phosphate dehydrogenase deficiency in Italian blood donors: prevalence and molecular defect characterization.

BACKGROUND: In the countries with high G6PD deficiency prevalence, blood donors are not routinely screened for this genetic defect. G6PD deficiency is often asymptomatic, blood donors may be carriers of the deficiency without being aware of it. The aim of the study was to evaluate the prevalence of G6PD deficiency among the Italian blood donors. DESIGN AND METHODS: From October 2009 to April 2011, 3004 blood donors from a large hospital transfusion centre were screened for G6PD deficiency using differential pH-metry and the characterization of G6PD mutations was performed on G6PD-deficient subjects. The haematological features of G6PD-deficient and normal donors were also compared. RESULTS: Thirty-three subjects (25 men and 8 women) with low G6PD activity were identified, corresponding to 1·1% of the examined blood donor population. The frequencies of class II severe alleles (Mediterranean, Valladolid, Chatham and Cassano) and class III mild alleles (Seattle, A- and Neapolis) were 48% and 43%, respectively. The haematological parameters of G6PD- donors were within normal range; however, the comparison between normal and G6PD- class II donors showed significant differences. CONCLUSION: In Italy, the presence of blood donors with G6PD deficiency is not a rare event and the class II severe variants are frequent. The identification of G6PD-deficient donors and the characterization of the molecular variants would prevent the use of G6PD-deficient RBC units when the haemolytic complications could be relevant especially for high risk patients as premature infants and neonates and patients with sickle cell disease submitted to multiple transfusions.

Hyperion's sponge-like appearance.

Hyperion is Saturn's largest known irregularly shaped satellite and the only moon observed to undergo chaotic rotation. Previous work has identified Hyperion's surface as distinct from other small icy objects but left the causes unsettled. Here we report high-resolution images that reveal a unique sponge-like appearance at scales of a few kilometres. Mapping shows a high surface density of relatively well-preserved craters two to ten kilometres across. We have also determined Hyperion's size and mass, and calculated the mean density as 544 +/- 50 kg m(-3), which indicates a porosity of >40 per cent. The high porosity may enhance preservation of craters by minimizing the amount of ejecta produced or retained, and accordingly may be the crucial factor in crafting this unusual surface.

A test of general relativity using radio links with the Cassini spacecraft.

According to general relativity, photons are deflected and delayed by the curvature of space-time produced by any mass. The bending and delay are proportional to gamma + 1, where the parameter gamma is unity in general relativity but zero in the newtonian model of gravity. The quantity gamma - 1 measures the degree to which gravity is not a purely geometric effect and is affected by other fields; such fields may have strongly influenced the early Universe, but would have now weakened so as to produce tiny--but still detectable--effects. Several experiments have confirmed to an accuracy of approximately 0.1% the predictions for the deflection and delay of photons produced by the Sun. Here we report a measurement of the frequency shift of radio photons to and from the Cassini spacecraft as they passed near the Sun. Our result, gamma = 1 + (2.1 +/- 2.3) x 10(-5), agrees with the predictions of standard general relativity with a sensitivity that approaches the level at which, theoretically, deviations are expected in some cosmological models.

Measurement and implications of Saturn's gravity field and ring mass.

The interior structure of Saturn, the depth of its winds, and the mass and age of its rings constrain its formation and evolution. In the final phase of the Cassini mission, the spacecraft dived between the planet and its innermost ring, at altitudes of 2600 to 3900 kilometers above the cloud tops. During six of these crossings, a radio link with Earth was monitored to determine the gravitational field of the planet and the mass of its rings. We find that Saturn's gravity deviates from theoretical expectations and requires differential rotation of the atmosphere extending to a depth of at least 9000 kilometers. The total mass of the rings is (1.54 ± 0.49) × 1019 kilograms (0.41 ± 0.13 times that of the moon Mimas), indicating that the rings may have formed 107 to 108 years ago.

Selective coupling of HE11 and TM01 modes into microfabricated fully metal-coated quartz probes.

We report computational and experimental investigations on injection and transmission of light in microfabricated fully Aluminum-coated quartz probes. In particular, we show that a selective coupling of either the HE(11) or the TM(01) mode can be carried out by injecting focused linearly or radially polarized beams into the probe. Optical fields, emitted by the probe after a controlled injection, are characterized in intensity and phase with the help of an interferometric technique. With the help of near-field measurement, we finally demonstrate that a longitudinally polarized spot localized at the tip apex is actually produced when the TM(01) mode is coupled into the probe.

Occurrence of two phosphorylated forms of yeast fructose-1,6-bisphosphatase with different isoelectric points.

Yeast fructose-1,6-bisphosphatase (EC 3.1.3.11) immunoprecipitated from glucose-derepressed wild-type cells and subjected to isoelectric focusing, appears as a unique peak, essentially homogeneous and devoid of incorporated phosphate. However, after cell incubation with glucose, two phosphorylated forms are detectable. The isoelectric point of one is higher and of the other is lower than that of the native form. In contrast, in the mutant ABYS1 which is deficient in several vacuolar proteinases (Achstetter, T., Emter, O., Ehmann, C. and Wolf, D.H. (1984) J. Biol. Chem. 259, 13334-13343), only the more acidic phospho form appears after cell incubation with glucose. However, sequence data rule out the possibility that limited proteolysis is the event responsible for the appearance of the more basic form of the phosphoenzyme. Nevertheless, time courses of glucose-induced inactivation of fructose-1,6-bisphosphatase show that the enzyme undergoes a substantially slower inactivation in the ABYS1 mutant as compared to the wild-type. These findings point to a degradative mechanism involving, besides the well-known phosphorylation, an additional as yet unknown modification which probably sensitizes the enzyme to proteolytic attack; furthermore, the enzyme responsible for such a modification seems to require one or more of the vacuolar proteinases missing in the mutant for its maturation.

Identification of a phosphorylated form of phosphoenolpyruvate carboxykinase from the yeast Saccharomyces cerevisiae.

A phosphoprotein of 65 kDa, as determined by SDS-gel electrophoresis, has been isolated from yeast crude extracts. This phospho form copurifies with phosphoenolpyruvate carboxykinase in the enzyme purification procedure worked out in our laboratory (Tortora, P., Hanozet, G.M. and Guerritore, A. (1985) Anal. Biochem. 144, 179-185). Moreover, both proteins bind strongly to 5'AMP-Sepharose 4B in the presence of Mn2+, whereas a substantially lower binding occurs if Mn2+ is replaced by Mg2+. This binding pattern is consistent with the well-known Mn2+-dependence of yeast phosphoenolpyruvate carboxykinase. These data suggest that the 65-kDa protein might be a phosphorylation product of the native enzyme. Furthermore, although the phospho form is not immunoprecipitated by anti-phosphoenolpyruvate carboxykinase antibodies, addition of Protein A-Sepharose CL-4B to crude extracts preincubated with the antibodies results in the binding to the resin of the phospho form, thus providing immunological evidence for its identification as a modified form of native enzyme. The same 65-kDa phosphoprotein is detectable in extracts from cells grown in the presence of [32P]Pi, as well as in cell extracts incubated with [gamma-32P]ATP. Moreover, digestion of the phosphoprotein with BrCN or with Staphylococcus aureus V8 proteinase, yields two and three fragments, respectively, which appear parallel to digestion products of phosphoenolpyruvate carboxykinase, again supporting the proposed identification. Finally, analysis of the phosphorylated amino acids in the 65-kDa protein shows that phosphoserine is the only labelled phosphoamino acid.

A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus.

A proteinase was purified to electrophoretic homogeneity from crude extracts of the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular mass values assessed by SDS-PAGE and gel filtration were 54 and 118 kDa, respectively, which points to a dimeric structure of the molecule. An isoelectric point of 5.6 was also determined. The enzyme behaved as a chymotrypsin-like serine proteinase, as shown by the inhibitory effects exerted by phenylmethanesulfonyl fluoride, 3,4-dichloroisocoumarin, tosylphenylalaninechloromethyl ketone and chymostatin. Consistently with the inhibition pattern, the enzyme cleaved chromogenic substrates at the carboxyl side of aromatic or bulky aliphatic amino acids; however, it effectively attacked only a small number of such substrates, thus, displaying a specificity much narrower than and clearly different from that of chymotrypsin. This was confirmed by its inability to digest a set of natural substrate proteins, as well as insulin chains A and B; only after alkylation casein was degraded to some extent. Proteinase activity was significantly stimulated by Mn2+ which acted as a mixed-type nonessential activator. The enzyme also displayed a broad pH optimum in the range 6.5-8.0. Furthermore, it was completely stable up to 90 degrees C; above this temperature it underwent first-order thermal inactivation with half-lives ranging from 342 min (92 degrees C) to 7 min (101 degrees C). At 50 degrees C it could withstand 6 M urea and, to some extent, different organic solvents; however, at 95 degrees C it was extensively inactivated by all of these compounds. None of the chemical physical properties of the enzyme, including amino-acid analysis, provided evidence of a possible relation to other well-known microbial serine proteinases.

Studies on the degradative mechanism of phosphoenolpyruvate carboxykinase from yeast Saccharomyces cerevisiae.

Previous work carried out in our laboratory (Burlini, N., Lamponi S., Radrizzani, M., Monti, E. and Tortora P. (1987) Biochim. Biophys. Acta 930, 220-229) led to the immunological identification of a yeast 65-kDa phosphoprotein as a modified form of phosphoenolpyruvate carboxykinase; moreover the appearance of this phospho form was proven to be independent of cAMP, whereas the glucose-induced inactivation of the native enzyme is cAMP-dependent. Here, we report further investigations on the mechanism of the glucose-triggered degradation of the enzyme which led to the following results: (a) the aforementioned phospho form displayed a binding pattern to 5 AMP-Sepharose 4B quite similar to that of native enzyme, although it did not retain its oligomeric structure, nor was it catalytically active; (b) its phosphate content was of about two residues per monomer; (c) its isoelectric point was slightly higher than that of native enzyme, this shows that the enzyme undergoes additional modifications besides phosphorylation; (d) it represented about 4% of the native enzyme in glucose-depressed cells; (e) other forms immunologically cross-reactive with the native enzyme were also isolated, whose molecular mass was in the range of 60-62 kDa, and they are probable candidates as degradation products of the phospho form; (f) time courses of the native and phospho forms in the presence and the absence of glucose provided data consistent with a kinetic model involving a strong stimulation of the decay of both forms effected by the sugar; (g) in the mutant ABYS1 (Achstetter, T., Emter, O., Ehmann, C. and Wolf, D.H. (1984) J. Biol. Chem. 259, 13334-13343) which is devoid of the four major vacuolar proteinases, the decay pattern was essentially the same as in wild-type; (h) effectors lowering intracellular ATP also retarded the first step of enzyme degradation; this points to an ATP-dependence of this step. Based on these results we propose a degradation mechanism consisting of an initial cAMP- and ATP-dependent modification of the enzyme, followed by a cAMP-independent phosphorylation, which leads to the appearance of the aforementioned monomeric phospho form; this in turn seems to undergo limited proteolysis. These data strongly suggest the occurrence of an intermediate form arising from the native one and whose phosphorylation gives rise to the 65-kDa phosphoprotein described here.

Selective denaturation of several yeast enzymes by free fatty acids.

The denaturation of eight purified yeast enzymes, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, alcohol dehydrogenase, beta-fructosidase, hexokinase and glucose-6-phosphate isomerase, promoted under controlled conditions by the free fatty acids myristic and oleic, is selective. Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1 oxidoreductase, EC 1.1.1.49) is extremely sensitive to destabilization and was studied in greater detail. Results show that chain length and degree of unsaturation of fatty acids are important to their destabilizing effect, and that ligands of the enzyme can afford protection. The denaturation process results in more than one altered form. These results can be viewed in the perspective of the possibility that amphipathic substances, and in particular free fatty acids, may play a role for enzyme degradation in vivo, by initiating steps of selective denaturation.

Susceptibility to proteinases of yeast enzymes selectively modified by fatty acids.

To investigate a possible correlation between selective modification and degradation of enzymes, the susceptibility to intracellular yeast proteinases A and B of yeast enzymes treated with fatty acids was tested. Enzymes used were glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 3-phosphoglycerate kinase (EC 2.7.2.3), which are sensitive to the denaturing modification caused by fatty acids, and alcohol dehydrogenase (EC 1.1.1.1) which is insensitive. Proteinases and substrate enzymes were all pure preparations. Without modification by fatty acids, at neutral pH, the three enzymes are remarkably resistant to degradation by both proteinases. Treatment with myristic or oleic acid definitely enhances the susceptibility to proteolysis of the sensitive glucose-6-phosphate dehydrogenase and 3-phosphoglycerate kinase, whereas it leaves negligible that of the insensitive alcohol dehydrogenase. The selective effect of fatty acids on the degradation is pH-dependent: with proteinase A it was lost at acidic pH. Since intracellular levels of free fatty acids near or even higher than 1 mM were actually measured in yeast cells, it is possible that free fatty acids, in some cellular conditions, affect yeast enzyme composition. However, the control of specific enzyme degradation in yeast is still an open question.

The powerful high pressure tool for protein conformational studies.

The pressure behavior of proteins may be summarized as a the pressure-induced disordering of their structures. This thermodynamic parameter has effects on proteins that are similar but not identical to those induced by temperature, the other thermodynamic parameter. Of particular importance are the intermolecular interactions that follow partial protein unfolding and that give rise to the formation of fibrils. Because some proteins do not form fibrils under pressure, these observations can be related to the shape of the stability diagram. Weak interactions which are differently affected by hydrostatic pressure or temperature play a determinant role in protein stability. Pressure acts on the 2 degrees, 3 degrees and 4 degrees structures of proteins which are maintained by electrostatic and hydrophobic interactions and by hydrogen bonds. We present some typical examples of how pressure affects the tertiary structure of proteins (the case of prion proteins), induces unfolding (ataxin), is a convenient tool to study enzyme dissociation (enolase), and provides arguments to understand the role of the partial volume of an enzyme (butyrylcholinesterase). This approach may have important implications for the understanding of the basic mechanism of protein diseases and for the development of preventive and therapeutic measures.

Various cells retrovirally transduced with N-acetylgalactosoamine-6-sulfate sulfatase correct Morquio skin fibroblasts in vitro.

Gene therapy may provide a long-term approach to the treatment of mucopolysaccharidoses. As a first step toward the development of an effective gene therapy for mucopolysaccharidosis type IVA (Morquio syndrome), a recombinant retroviral vector, LGSN, derived from the LXSN vector, containing a full-length human wildtype N-acetylgalactosamine-6-sulfate sulfatase (GALNS) cDNA, was produced. Severe Morquio and normal donor fibroblasts were transduced by LGSN. GALNS activity in both Morquio and normal transduced cells was several fold higher than normal values. To measure the variability of GALNS expression among different transduced cells, we transduced normal and Morquio lymphoblastoid B cells and PBLs, human keratinocytes, murine myoblasts C2C12, and rabbit synoviocytes HIG-82 with LGSN. In all cases, an increase of GALNS activity after transduction was measured. In Morquio cells co-cultivated with enzyme-deficient transduced cells, we demonstrated enzyme uptake and persistence of GALNS activity above normal levels for up to 6 days. The uptake was mannose-6-phosphate dependent. Furthermore, we achieved clear evidence that LGSN transduction of Morquio fibroblasts led to correction of the metabolic defect. These results provide the first evidence that GALNS may be delivered either locally or systematically by various cells in an ex vivo gene therapy of MPS IVA.

Expression of a synthetic gene encoding P2 ribonuclease from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus in mesophylic hosts.

This work reports the molecular cloning and expression of a synthetic gene encoding P2, a 7-kDa ribonuclease (RNase) previously isolated in our laboratory from the archaebacterium Sulfolobus solfataricus [Fusi et al., Eur. J. Biochem. 211 (1993) 305-310]. The P2-encoding synthetic gene was expressed in E. coli and in Saccharomyces cerevisiae. The recombinant (re-) protein was produced to approx. 1.5% of the total protein content in S. cerevisiae using the galactose-inducible GAL1 promoter and to 3% (tac/lac tandem promoters) or 6.5% (T7 promoter) in E. coli as judged by immunological and biochemical criteria. E. coli-produced P2 was purified to electrophoretic homogeneity through a one-step procedure, i.e., DEAE-Sephacel chromatography at pH 9.3. S. cerevisiae-produced P2 additionally required filtration through a Centricon-10 microconcentrator to obtain the same purity. The re-P2 was found to be indistinguishable from the Su. solfataricus enzyme on the basis of heat stability, pH optimum and RNA digestion pattern. Furthermore, monodimensional nuclear magnetic resonance showed that the E. coli- and Su. solfataricus-produced enzymes were structurally identical, the only exceptions being that Lys4 and Lys6 were not methylated in the re-enzyme, thus showing that lysine methylation does not play a role in P2 thermostabilization.

Glucose-induced degradation of yeast fructose-1,6-bisphosphatase requires additional triggering events besides protein phosphorylation.

Glucose addition to yeast cells stimulates a cAMP overshoot with concomitant activation of cAMP-dependent protein kinase, which in turn rapidly phosphorylates fructose-1,6-bisphosphatase. The phosphorylated enzyme subsequently undergoes a slow proteolytic breakdown. Also, it has been proposed that phosphorylation represents the mechanism that initiates proteolysis. Here we present experiments carried out on a yeast mutant defective in adenylate cyclase [(1982) Proc. Natl. Acad. Sci. USA 79, 2355-2359] in which extracellular cAMP triggers full enzyme phosphorylation but a scanty proteolysis, whereas glucose plus cAMP provoke both phosphorylation and complete proteolytic breakdown. Thus, besides a glucose-induced cAMP peak, which results in enzyme phosphorylation, other effects evoked by the sugar are indispensable for its proteolytic degradation.

Molecular cloning, nucleotide sequence and expression of a Sulfolobus solfataricus gene encoding a class II fumarase.

Fumarase catalyzes the interconversion of L-malate and fumarate. A Sulfolobus solfataricus fumarase gene (fumC) was cloned and sequenced. Typical archaebacterial regulatory sites were identified in the region flanking the fumC open reading frame. The fumC gene encodes a protein of 438 amino acids (47,899 Da) which shows several significant similarities with class II fumarases from both eubacterial and eukariotic sources as well as with aspartases. S. solfataricus fumarase expressed in Escherichia coli retains enzymatic activity and its thermostability is comparable to that of S. solfataricus purified enzyme despite a 11 amino acid C-terminal deletion.

Regulation of maltose utilization in Saccharomyces cerevisiae by genes of the RAS/protein kinase A pathway.

In Saccharomyces cerevisiae maltose utilization requires a functional MAL locus, each composed of three genes: MALR (gene 3) encoding a regulatory protein, MALT (gene 1) encoding maltose permease and MALS (gene 2) encoding maltase. We show that constitutive activation of the RAS/protein kinase A pathway severely reduces growth of MAL1 strains on maltose. This may be a consequence of reduction in MALT mRNA, reduced Vmax and increased catabolite inactivation of the MALT-encoded maltose transporter in the MAL1 strain. Mutations in the GGS1/TPS1 gene, which restricts glucose influx and possibly affects signalling, relieve carbon catabolite repression on both maltase and maltose permease and reduce maltose permease inactivation.

An 8.5-kDa ribonuclease from the extreme thermophilic archaebacterium Sulfolobus solfataricus.

Protein p3, a ribonuclease we previously isolated from the archaebacterium Sulfolobus solfataricus [P. Fusi et al. (1993) Eur. J. Biochem. 211, 305-310], was subjected to complete amino acid sequencing. It consisted of 75 residues, with a calculated M(r) of 8582, a pI of 10.1, and had some degree of monomethylation at Lys-4 and Lys-6. p2, a previously sequenced, 62-residue ribonuclease from the same organism, had an identical sequence for 57 consecutive residues starting from the N-terminus. p2 and p3 also showed a striking similarity to five other proteins previously isolated from Sulfolobus strains and identified as DNA-binding proteins. However, the C-terminus, 10 residue region of p3 did not show any similarity to these proteins; in contrast, it was significantly similar to stretches in three eubacterial ribonucleases from Bacillus strains. No difference between p2 and p3 has so far been detected as regards their catalytic properties. Available data suggest that these molecules have a narrow substrate specificity and probably play specific roles in RNA processing.