Paleogeographic reconstructions of coastal aegean archaeological sites.

Many studies have been made of ancient Greek topography, some of the more recent ones based on modern techniques. However, most still ignore the subsurface dimension of coastal and other environments and hence fail to fully explain coastal and alluvial-colluvial processes, rates of change of geomorphology, and the effects of coastal change on humans. In this article subsurface geological analyses have been used to elucidate paleogeographic coastal settings of major archaeological sites around the Aegean Sea. Similar approaches could be applied in the Middle and Far East and elsewhere in the Mediterranean.

Pumice from thera (santorini) identified from a greek mainland archeological excavation.

Pumice fragments recovered from an archeological excavation on the Greek mainland have been correlated by means of index of refraction measurements with the Late Bronze Age volcanic eruption on the island of Thera (Santorini) in the Aegean Sea. Pottery from the strata containing the pumice dates from the 15th century B.C.

Low amounts of PEG-lipid induce cubic phase in phosphatidylethanolamine dispersions.

By using time-resolved X-ray diffraction we demonstrate that low amounts (5-10 mol%) of a phospholipid with two saturated hydrocarbon acyl chains 14 carbon atoms long and PEG550 chain covalently attached to its phosphoethanolamine polar head group, DMPE(PEG550), induce spontaneous formation of a cubic phase with lattice constant 20.5 nm (cubic aspect #8, space group Im3m) in aqueous dispersions of dielaidoylphosphatidylethanolamine (DEPE). This phase displays a highly resolved X-ray diffraction pattern with 17 low-angle reflections. The cubic phase was found to intrude in the temperature range between the lamellar liquid crystalline (L(alpha)) phase and the inverted hexagonal phase (H(II)) known to form in pure DEPE/water dispersions. A higher DMPE(PEG550) amount of 20 mol% was found to eliminate the non-lamellar phases in the temperature scale up to 100 degrees C. DMPE grafted with PEG5000 only shifts the L(alpha)-H(II) transition of DEPE to higher temperatures but does not promote formation of cubic phase. These findings indicate that, consistent with their bulky head groups, the PEG-lipids decrease the tendency for negative interfacial mean curvature of the DEPE bilayers.

An ordered metastable phase in hydrated phosphatidylethanolamine: the Y-transition.

By using time-resolved X-ray diffraction, differential scanning calorimetry and scanning densitometry, we observed rapid formation at low temperature of a metastable ordered phase, termed LR1 phase, in fully hydrated dihexadecylphosphatidylethanolamine (DHPE). The LR1 phase has the same lamellar repeat period as the gel Lbeta phase but differs from the latter in its more ordered, orthorhombic hydrocarbon chain arrangement. It forms at about 12 degrees C upon cooling and manifests itself as splitting of the sharp, symmetric wide-angle X-ray peak of the DHPE gel phase into two reflections. This transition, designated the 'Y-transition', is readily reversible and proceeds with almost no hysteresis between cooling and heating scans. Calorimetrically, the LR1-->Lbeta transition is recorded as a low-enthalpy (0.2 kcal/mol) endothermic event. The formation of the LR1 phase from the gel phase is associated with a small, about 2 microl/g, decrease of the lipid partial specific volume recorded by scanning densitometry, in agreement with a volume calculation based on the X-ray data. The formation of the equilibrium Lc phase was found to take place from within the LR1 phase. This appears to be the only observable pathway for crystallisation of DHPE upon low-temperature incubation. Once formed, the Lc phase of this lipid converts directly into Lbeta phase at 50 degrees C, skipping the LR1 phase. Thus, the LR1 phase of DHPE can only be entered by cooling of the gel Lbeta phase. The data disclose certain similarities between the low-temperature polymorphism of DHPE and that of long-chain normal alkanes.

New phases induced by sucrose in saturated phosphatidylethanolamines: an expanded lamellar gel phase and a cubic phase.

A new lamellar gel phase (L beta *) with expanded lamellar period was found at low temperatures in dihexadecylphosphatidylethanolamine (DHPE) and dipalmitoylphosphatidylethanolamine (DPPE) dispersions in concentrated sucrose solutions (1-2.4 M). It forms via a cooperative, relatively broad transition upon cooling of the L beta gel phase of these lipids. According to the X-ray data, the transformation between L beta and L beta * is reversible, with a temperature hysteresis of 6-10 degrees C and a transition width of about 10 degrees C. No specific volume changes and a very small heat absorption of about 0.05 kcal/mol accompany this transition. The L beta *-L beta transition temperature strongly depends on the disaccharide concentration. From a value of about 10 degrees C below the melting transition of DHPE, it drops by 25 degrees C with decrease of sucrose concentration from 2.4 M to 1 M. The low-temperature gel phase L beta * has a repeat spacing by 8-10 A larger than that of the L beta gel phase and a single symmetric 4.2 A wide-angle peak. It has been observed in 1, 1.25, 1.5 and 2.4 M solutions of sucrose, but not in 0.5 M of sucrose. The data clearly indicate that the expanded lamellar period of the L beta * phase results from a cooperative, reversible with the temperature, increase of the interlamellar space of the L beta gel phase. Other sugars (trehalose, maltose, fructose, glucose) induce similar expanded low-temperature gel phases in DHPE and DPPE. The L beta * phase is osmotically insensitive. Its lamellar period does not depend on the sucrose concentration, while the lattice spacings of the L alpha, L beta and HII phases decrease linearly with increase of sucrose concentration. Another notable sugar effect is the induction of a cubic phase in these lipids. It forms during the reverse HII-L alpha transition and coexists with the L alpha phase in the whole temperature range between the HII and L beta phases. The cubic phase has only been observed at sucrose concentrations of I M and above. In accordance with previous data, sucrose suppresses the L alpha phase in both lipids and brings about a direct L beta-HII phase transition in DHPE. A raid, reversible gel-subgel transformation takes place at 17 degrees C in both DPPE and DHPE. Its properties do not depend on the sucrose concentration. The observed new effects of disaccharides on the properties of lipid dispersions might be relevant to their action as natural protectants.

Time-resolved cryo-electron microscopic study of the dissociation of actomyosin induced by photolysis of photolabile nucleotides.

The rapid release of a substrate or other ligand from photolabile precursors in a thin layer suspension of biological specimens followed by rapid freezing provides a method of trapping and visualizing short-lived states in a dynamic system. We demonstrate here the first successful application of this method to study the interaction of actin filaments with myosin subfragment 1 (S1) after release of nucleotides. The results obtained suggest that structural changes in actin filaments occur as a result of interaction with S1.

Submillisecond changes in myosin lattice spacing resulting from rapid length changes.

The speed of the myofilament lattice spacing response to rapid changes in load or length of single, intact muscle fibres of the frog, was investigated during isometric tetani. During ramp releases at close to Vmax and during step length changes (completed within 250 microseconds), lattice spacing was calculated from the equatorial X-ray diffraction pattern (sampled at 250 microseconds time resolution using synchrotron radiation). Ramp releases (total shortening=1.39 %) caused a spacing increase, described with an exponential function (alpha=271 s-1, amplitude=1.15 nm) plus an elastic component having the time course of discharge of axial tension (amplitude 0.28 nm). For a step release (amplitude=0.87%), lattice expansion could be described with an exponential (alpha =1005 s-1, amplitude=0.56 nm) plus an elastic component of 0.25 nm amplitude. Lattice compression was associated with a step stretch (amplitude=0.62 %), and was also quasi-exponential (alpha=367 s-1, amplitude=0.74 nm), with an elastic component of 0.28 nm. The spacing change time course for length steps resembled that of the accompanying quick recovery of axial tension and the associated change in the meridional 14.5 nm reflection intensity, which are both believed to be determined by the kinetics of the molecular power stroke. Therefore, this shows that lattice spacing changes, arising from radial forces exerted by attached crossbridges, are fast enough to occur during the power stroke event.

Time-resolved equatorial X-ray diffraction studies of skinned muscle fibres during stretch and release.

Equatorial X-ray diffraction patterns were recorded from small bundles of one to three chemically skinned frog sartorius muscle fibres (time resolution 250 microseconds) during rapid stretch and subsequent release. In the relaxed state, the dynamic A-band lattice spacing change as a result of a 2 % step stretch (determined from the positions of the 10 and 11 reflections) resulted in a 21 % increase in lattice volume, while static studies of spacing and sarcomere length indicated than an increase in volume of >/=50 % for the same length change. In rigor, stretch caused a lattice volume decrease which was reversed by a subsequent release. In activated fibres (pCa 4.5) exposed to 10 mM 2,3-butanedione 2-monoxime (BDM), stretch was accompanied by a lattice compression exceeding that of constant volume behaviour, but during tension recovery, compression was partially reversed to leave a net spacing change close to that observed in the relaxed fibre. In the relaxed state, spacing changes were correlated with the amplitude of the length step, while in rigor and BDM states, spacing changes correlated more closely with axial force. This behaviour is explicable in terms of two components of radial force, one due to structural constraints as seen in the relaxed state, and an additional component arising from cross-bridge formation. The ratio of axial to radial force for a single thick filament resulting from a length step was four in rigor and BDM, but close to unity for the relaxed state.

Structural characterization of the pressure-denatured state and unfolding/refolding kinetics of staphylococcal nuclease by synchrotron small-angle X-ray scattering and Fourier-transform infrared spectroscopy.

The pressure-induced unfolding of wild-type staphylococcal nuclease (Snase WT) was studied using synchrotron X-ray small-angle scattering (SAXS) and Fourier-transform infrared (FT-IR) spectroscopy, which monitor changes in the tertiary and secondary structural properties of the protein upon pressurization. The experimental results reveal that application of high-pressure up to 3 kbar leads to an approximate twofold increase of the radius of gyration Rg of the native protein (Rg approximately 17 A) and a large broadening of the pair-distance-distribution function, indicating a transition from a globular to an ellipsoidal or extended chain structure. Analysis of the FT-IR amide I' spectral components reveals that the pressure-induced denaturation process sets in at 1.5 kbar at 25 degrees C and is accompanied by an increase in disordered and turn structures while the content of beta-sheets and alpha-helices drastically decreases. The pressure-induced denatured state above 3 kbar retains nonetheless some degree of beta-like secondary structure and the molecule cannot be described as a fully extended random coil. Temperature-induced denaturation involves a further unfolding of the protein molecule which is indicated by a larger Rg value and significantly lower fractional intensities of IR-bands associated with secondary-structure elements. In addition, we have carried out pressure-jump kinetics studies of the secondary-structural evolution and the degree of compactness in the folding/unfolding reactions of Snase. The effect of pressure on the kinetics arises from a larger positive activation volume for folding than for unfolding, and leads to a significant slowing down of the folding rate with increasing pressure. Moreover, the system becomes two-state under pressure. These properties make it ideal for probing multiple order parameters in order to compare the kinetics of changes in secondary structure by pressure-jump FT-IR and chain collapse by pressure-jump SAXS. After a pressure jump from 1 bar to 2.4 kbar at 20 degrees C, the radius of gyration increases in a first-order manner from 17 A to 22.4 A over a timescale of approximately 30 minutes. The increase in Rg value is caused by the formation of an extended (ellipsoidal) structure as indicated by the corresponding pair-distance-distribution function. Pressure-jump FT-IR studies reveal that the reversible first order changes in beta-sheet, alpha-helical and random structure occur on the same slow timescale as that observed for the scattering curves and for fluorescence. These studies indicate that the changes in secondary structure and chain compactness in the folding/unfolding reactions of Snase are probably dependent upon the same rate-limiting step as changes in tertiary structure.

Giant vesicles from 72-membered macrocyclic archaeal phospholipid analogues: initiation of vesicle formation by molecular recognition between membrane components

Stereochemically pure archeal acyclic bola-amphiphilic diphosphates 4 and 5, with the basic structure of the phospholipids found in Sulfolobus, have been synthesized for the first time. The self-assembly properties have been compared with those of the nearly identical 72-membered macrocyclic tetraether phosphates 3a and 3b, analogues of the major phospholipid components of Sulfolobus, Thermoplasma, and methanogenic Archea, which were also synthesized. Phase contrast and fluorescence microscopies have shown that the dipolar lipids 1 and 2 spontaneously formed vesicles. Whereas the macrocyclic dipolar phosphates 3 spontaneously formed vesicles (phase contrast and fluorescence microscopies), the bolaform phosphate 4 gave only a lamellar structure (synchrotron diffraction pattern: repeat distance of about 4.25 nm but with only a few layers). However, upon addition of the unphosphorylated precursors phytanol, phytol, or geranylgeraniol to the acyclic lipids 4 and 5, giant vesicles were rapidly formed. Addition of n-hexadecanol or cholesterol did not lead to vesicle formation. Therefore it was concluded that this vesicle formation occurs only when the added molecule is closely compatible with the constituents of the lipid layer and can be inserted into the double layer. A slight mismatch (cholesterol or n-hexadecanol/polyprenyl chains) is therefore enough to block the insertion process presumably required for vesicle formation.

Characterization of three abundant mRNAs from human ovarian granulosa cells.

Three cDNA clones, pHGR122, pHGR11, and pHGR74 containing the coding information for abundant mRNAs were identified from a human ovarian granulosa cell cDNA library. Characterization by nucleotide sequencing revealed that pHGR122 was specific for a collagenase inhibitor and pHGR11 for melanoma-associated antigen ME491. Relative quantification by Northern analysis indicated that collagenase inhibitor mRNA is a major species in granulosa cells. This finding provides evidence for the origin of this protein in follicular fluid as a secretory product of granulosa cells. pHGR11 identified melanoma-associated antigen ME491 as the unexpected product of normal, noncarcinogenic, granulosa cells. pHGR74 has the complete coding information for an unknown protein. Three independent experiments: (i) cell-free translation of pHGR74 RNA; (ii) transcription of suitable restriction fragments followed by cell-free translation; (iii) hydrolysis of the cell-free translation product of pHGR74 RNA by endoproteinase Lys-C, identified one open reading frame coding for an acidic, highly hydrophilic protein of 111 amino acid residues. pHGR74 mRNA is expressed in human testis, prostate, seminal vesicle, and ovarian granulosa cells. A comparative Southern analysis indicates pHGR74 mRNA is species specific and encoded by a single-copy gene.

Composition and mechanical properties of gutta-percha endodontic points.

Gutta-percha endodontic filling points were found to contain approximately 20% gutta-percha (matrix), 66% zinc oxide (filler), 11% heavy metal sulfates (radiopacifier), and 3% waxes and/or resins (plasticizer). The mechanical properties were indicative of a partially crystalline viscoelastic polymeric material. They were found to obey Hooke's law and displayed a prominent upper and lower yield point when stressed beyond the proportional limit. The essential differences in mechanical properties of individual brands were found to be a function of the gutta-percha and zinc oxide concentration.

Enthalpy is a proper criterion for comparability of monolayer and bilayer studies: isobaric temperature scanning measurements on glycolipid monolayers.

The thermotropic behaviour of glycolipid monolayers has been studied by isobaric temperature scanning measurements to elucidate conditions under which monolayers exhibit thermodynamic and structural properties comparable to those observed in bilayers. A selection of synthetic, stereochemically pure, glyceroglycolipids with identical, ether-linked alkyl chains of 12, 14, or 16 CH2-groups has been investigated. The head groups of the glycolipids consisted of glucose, galactose, maltose, lactose or maltotriose moieties with beta-configuration of the glycosidic bond. These glycolipids were chosen to permit a quantitative characterization of three effects, (i) the role of the length of the aliphatic chains, (ii) the influence of the size of the head group, and (iii) the influence of the stereochemistry of the sugar moieties on the structure and stability of the monolayers. To probe the effects of stereochemical alterations in the glycerol moiety 2,3-O-ditetradecyl-1-O-beta-D-glucosyl-sn-glycerol (14-2,3-Glc) was compared with 1,2-O-ditetradecyl-3-O-beta-D-glucosyl-sn-glycerol (14-1,2-Glc). It has been shown that in general several features of bilayers can be obtained from monolayer studies with reasonable accuracy, provided the proper parameters are chosen. The monolayer is stabilized by elongation of the aliphatic chains of the lipids and destabilized when the monosaccharide read groups is replaced by a di-, or trisaccharide, in a similar manner as in the bilayer. The stabilizing effect that has been observed in bilayer studies, when galactose instead of glucose is introduced as head group, has also been established in the monolayer studies. This stabilizing effect is even retained in the lipids having disaccharide head groups. On the basis of these monolayer studies in connection with WAXS and SAXS measurements on multilamellar systems, we suggest that identity of transition enthalpies of the chain melting L beta-L alpha transition is an appropriate criterion for estimating molecular areas and area changes of bilayers from monolayer measurements and vice versa. However, estimates of transition temperatures are poor using the enthalpy criterion. If identity of transition temperature is introduced as criterion, glycolipid monolayers must be compressed to about 43 +/- 3 mN m-1. Under these conditions the agreement between the calculated enthalpies and structural properties of monolayers and multilayers is poor. As a general conclusion it can be emphasized that for monolayer and bilayer systems of glycolipids there exists no such parameter as a universal pressure or a universal temperature that automatically renders monolayer data identical to bilayer data. Depending on which property (transition temperatures, transition enthalpies, lateral areas and transitional area changes) one wants to extrapolate from monolayer to bilayer different lateral pressures have to be applied.

Comparison of two in vitro methods of bone lead analysis and the implications for in vivo measurements.

Atomic absorption spectrometry and x-ray fluorescence have been used to determine the lead content of metatarsal and tibia bone samples. For a range of bone lead levels from 6.5 to 83 micrograms g-1 of ashed bone there is no evidence of a systematic difference between the two techniques of more than 1 microgram g-1. There is, however, some evidence that random differences between the two in vitro analyses applied to the same bone sample are larger than can be accounted for by known measurement uncertainties. Variations in bone composition could account for these differences. Because the x-ray fluorescence technique is applied in an identical way to in vivo analysis, it is concluded that the uncertainties in in vivo measurements are small.

Idiopathic scoliosis: transcutaneous muscle stimulation versus the Milwaukee brace.

This paper compared 3-year results of electrical stimulation with the Milwaukee brace for the treatment of idiopathic scoliosis. Fifty patients in each group were compared retrospectively and matched for age, sex, Risser sign, and curve morphology. Evaluations were performed at 6-month intervals with radiographs and examinations. Skin irritation was the most common complication with electrical stimulation. Using survivorship analysis methods, no significant differences were found in rates of curve progression or failure. Overall, 70% of the patients in each group were successfully maintained over a course of 3 years. Electrical stimulation is comparable to the Milwaukee brace in managing idiopathic scoliosis.

Trampoline injuries.

We feel that there are dangers on the trampoline if it is poorly supervised. This is especially true in regards to the possibility of spinal cord injury and quadriplegia. We have presented documented cases of quadriplegia and safety suggestions have been given for the use of the teampoline. We hope that by presenting this problem, physicians, who are associated with the prevention and care of athletic injuries, will be made aware of the possibility of the dreaded quadriplegia associated with trampoline injuries and that this injury will be greatly decreased.

Miscibility gap in fluid dimyristoylphosphatidylcholine:cholesterol as "seen" by x rays.

A binary mixture of dimyristoylphosphatidylcholine (DMPC) and cholesterol displays a fluid miscibility gap under excess water conditions. Effects due to the imperfect miscibility of the two amphiphiles are studied near to and far from thermodynamic equilibrium by time-resolved small angle x-ray diffraction. The experiment discloses that this mixture phase separates when leaving the miscibility gap upon heating, a transition that is not included in current phase diagrams. This transition appears to be reversible and shows a temperature hysteresis of only a few degrees. We suggest a model in which the transition is driven with increasing temperature by a movement of the cholesterol away from the hydrophilic-hydrophobic interface toward the hydrophobic core of the bilayer.

The time course of changes in the equatorial diffraction patterns from different muscle types on photolysis of caged-ATP.

Using the synchrotron X-ray source at DESY, Hamburg, we have measured the time courses of changes in the strongest equatorial reflections from small bundles of chemically skinned fibres from insect flight muscle, Limulus muscle and rabbit psoas and soleus muscles following the photolytic release of ATP. In all preparations the release of ca. 2 mM ATP caused the tension to relax with a complex time course, the final relaxation rates in insect and rabbit psoas fibres being ca. 10 times faster than those measured in rabbit soleus and Limulus fibres (ca. 50 ms cf. ca. 500 ms half times). However, in all fibre types there was a very rapid change in equatorial intensities towards relaxed values (half time less than 5 ms), the extent of this change and the occurrence of a slower phase of intensity change being dependent on the preparation. In insect the 1.0 and 2.0 intensities change rapidly to their relaxed values; in Limulus the 1.0 and 1.1 intensities change rapidly to within ca. 10% of their relaxed values; in rabbit psoas the initial, rapid 1.1 intensity fall is to within 30-40% of its relaxed value and is followed by a slower fall which appears to correlate with the rate of the final tension relaxation, e.g. phosphate ions accelerate both rates; in rabbit soleus the equatorial response is very similar to that of psoas fibres. These results are discussed in terms of the model of Goldman et al. in which a rapid ATP induced dissociation of rigor crossbridges is followed by the transient cooperative reattachment of some bridges which may proceed through at least part of the cross-bridge cycle.

Studies on the 14.5 nm meridional X-ray diffraction reflection during length changes of intact frog muscle fibres.

The intensity of the 14.5 nm meridional reflection (M3) from activated skeletal muscle fibres was studied in both single fibres and fibre bundles during the imposition of length changes. During shortening at small load, the intensity of the reflection decreased within 2 ms to less than 20% of isometric intensity, then recovered partially during the remainder of the shortening. When shortening was terminated, recovery of intensity was delayed. Small shortening steps (0.5% fibre length) produced a fall in M3 intensity (IM3) delayed by ca. 250 microseconds compared to the fall in tension. For larger step releases (1% fibre length), the fall in IM3 was not delayed. The fall in IM3 could be almost completely reversed by a subsequent restretch applied within 1.5 ms. Beyond 10 ms after the initial release, the restretch caused a further fall in intensity. A rapid step stretch (0.5% fibre length) also caused a fall in IM3 without delay, which was partially reversed by a release applied within 10 ms. A second small release applied 3 ms (or less) after the first caused a second fall in M3 intensity, but without delay and with faster time course. Small amplitude sinusoidal length oscillations (0.15-0.2% sarcomere; 1 kHz) caused a sinusoidal change in M3 intensity, which was 180 degrees out of phase with the force oscillations, and lacked distortion during its release phase.

X-ray diffraction studies of the cross-bridge intermediate states.

Two dimensional x-ray diffraction was obtained from skinned rabbit psoas muscle fibers. The goal is to correlate structures of the cross-bridge population with various intermediate states in the cross-bridge cycle by using nucleotides and their analogs. It was found that in a relaxed muscle in ATP containing solutions, cross-bridges are distributed in three populations in equilibrium: those detached from actin and ordered on the myosin helix, those that are detached and disordered, and those weakly attached to actin in random orientations. The distribution among the three populations is highly dependent on temperature. Those that are detached and yet ordered in a helical structure surrounding the myosin backbone are very likely in the M.ADP.Pi state, supporting an earlier suggestion by Wray (1987). It was also found that the attached cross-bridges with bound MgADP are structurally distinct from those without nucleotide, in agreement with one of our earlier findings by osmotic compression (Xu et al., 1993). Another finding of interest is that the analog AMP-PNP was found to be an ATP analog, rather than an ADP analog as it has been reported previously by many research groups.