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PubMed1994

Protein crystallization in space.

The microgravity environment of space is an ideal place to study the complicated protein crystallization process and to grow good-quality protein crystals. A series of crystal growth experiments of 10 different proteins was carried out in space on a Chinese re-entry satellite FSW-2 in August, 1992. The experiments were performed for about two weeks at a temperature of 18.5 +/- 0.5 degrees C using a tube-like crystallization apparatus made in the Shanghai Institute of Technical Physics, Academia Sinica. More than half of 48 samples from 6 proteins produced crystals, and the effects of microgravity on protein crystal growth were observed, especially for hen-egg white lysozyme and an acidic phospholipase A2 from the venom of Agkistrodon halys Pallas. Analyses of the crystallization of these two enzymes in this mission showed that the microgravity environment in space may be beneficial to improve size, external perfection, morphology, internal order, and nucleation of protein crystals. Some of these positive microgravity effects were also demonstrated by the growth of protein crystals in gelled solution with the above two enzymes. A structural analysis of the tetragonal lysozyme crystal grown in space is in progress.

Ancrod↗
PubMed1991

Inclusion of proteins into calcium oxalate crystals precipitated from human urine: a highly selective phenomenon.

The abundance of protein in the matrix of calcium oxalate uroliths has fueled speculation regarding its role in stone genesis. In this study, we wanted to characterize the composition of the proteins associated with early stages of calcium oxalate crystallization in urine. Calcium oxalate crystallization was induced in urine from healthy men and women by the addition of an oxalate load. The crystals were harvested and demineralized, and the proteins remaining were separated and characterized by polyacrylamide gel electrophoresis and Western blotting. Most urinary proteins were not detected in the crystals or were present in only small quantities. The most abundant urinary macromolecule, Tamm-Horsfall glycoprotein, was notably absent from the crystal extracts. The predominant protein associated with the crystals, a previously unknown urinary constituent that we call crystal matrix protein (CMP; molecular mass, 30,000 Da), was more prevalent in the crystals derived from female urine. We conclude that most urinary proteins play no direct role in calcium oxalate crystal formation. However, the protein CMP exhibits a remarkable affinity for calcium oxalate crystals and may be important in stone pathogenesis.

Blotting, Western↗
PubMed1990

Glycosaminoglycans alter the capacity of low density lipoprotein to bind to monosodium urate crystals.

Low density lipoprotein (LDL) has previously been demonstrated to be a potent inhibitor of human inflammatory cell activation by monosodium urate (MSU) crystals in vitro. As suppression of cellular responses to crystals by LDL is known to be dependent on the binding of LDL to urate crystals we further evaluated the mechanism and regulation of LDL binding to urate crystals in vitro. Using nonlinear, least squares methodology to analyze binding, we found LDL to saturably and reversibly bind with high affinity (Kd 9.3 x 10(-9) M) to MSU crystals. LDL binding was competitively inhibited by LDL but not by a variety of other positively charged moieties. Glycosaminoglycans (GAG) (heparin, heparan sulfate, hyaluronate and chondroitin sulfate), in diminishing order of potency, inhibited the binding of LDL to crystals. This inhibitory activity was dose dependent, sensitive to digestion with glycosidases and appeared to be specific for polymerized GAG, as glucuronic acid, dextran, dextran sulfate, as well as isolated amino sugar constituents of GAG were either weakly inhibitory or inactive. GAG also promoted dissociation of bound LDL from the urate crystal surface. The results indicate that LDL binds saturably and reversibly to urate crystals and that polymerized, but not depolymerized, hyaluronate and other GAG inhibit LDL binding to urate crystals. This suggests that the amount of LDL coating intraarticular urate crystals could vary during the course of a gouty paroxysm.

Crystallization↗
PubMed1986

Purification and crystallization of creatine kinase from rabbit skeletal muscle.

Crystallization is the primary rate-limiting step in protein structure determination. It has been our experience over approximately 10 years that crystals are obtained in about 20% of the proteins attempted and that only about 10% of these crystals are sufficiently well ordered to permit atomic resolution structure analysis. In attempts to overcome this limitation, we have investigated the effect on crystallization of microheterogeneity in a protein regarded as pure by conventional criteria. Creatine kinase was purified from rabbit skeletal muscle and crystallized from methylpentanediol. The protein appeared to be nearly pure judging by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high specific activity. The crystals that were obtained were of poor quality, and an extensive survey of precipitants, crystallization conditions, and additives failed to discover conditions from which usable crystals could be obtained. The enzyme was then subjected to a series of further purification steps. After each purification step, the quality of the crystals obtained under almost identical conditions improved. The final purification step was flat-bed isoelectric focusing. Crystals grown from focused creatine kinase are well ordered and diffract to approximately 3-A resolution.

Animals↗
PubMed1994

Urinary glycosaminoglycans are selectively included into calcium oxalate crystals precipitated from whole human urine.

Urinary glycosaminoglycans are selectively included into calcium oxalate (CaOx) crystals precipitated from whole human urine: The presence of glycosaminoglycans (GAGs) in the organic matrix of urinary stones, and their known effects on CaOx crystallization have prompted speculation regarding their role in CaOx urolithiasis. The aim of this study was to examine the involvement of GAGs in the early stages of CaOx crystallization in human urine. Urine samples were collected from healthy men and CaOx crystallization was induced by the addition of a sodium oxalate load. The crystals were harvested and demineralized, and the GAG content of the resulting extract analysed by cellulose acetate electrophoresis. Only one GAG, heparan sulphate (HS) was detected in the organic matrix of the crystals; chondroitin sulphate (ChS), the most abundant urinary GAG, was conspicuously absent. Further experiments, in which varying amounts of HS and ChS were added to ultrafiltered (10,000 Da) urine prior to induction of calcium oxalate crystallization, showed that ChS was included into the crystals only when HS was absent from the urine. It was concluded that the selective inclusion of GAGs into crystals and stones is a function related more to relative binding affinity than to ambient GAG concentration and that HS and ChS compete for specific binding sites on the crystal surface.

Adult↗
PubMed1994

Protein crystal growth in microgravity.

Protein crystal growth is quite important for the determination of protein structures which are essential to the understanding of life at molecular level as well as to the development of molecular biotechnology. The microgravity environment of space is an ideal place to study the complicated protein crystallization and to grow good-quality protein crystals. A number of crystal-growth experiments of 10 different proteins were carried out in August, 1992 on the Chinese re-entry satellite FSW-2 in space using a tube crystallization equipment made in China. A total of 25 samples from 6 proteins produced crystals, and the effects of microgravity on protein crystal growth were observed, especially for an acidic phospholipase A2 and henegg-white lysozyme which gave better crystals in space than earth-grown crystals in ground control experiments. The results have shown that the microgravity in space favors the improvement of the size, perfection, morphology and internal order of the grown protein crystals.

Crystallization↗
PubMed1994

Effects of Tamm-Horsfall glycoprotein and albumin on struvite crystal growth in urine of cats.

Tamm-Horsfall protein (THP) was isolated and purified, using pooled urine collections from clinically normal cats. The effects of feline THP and purified feline serum albumin on urine chemical and struvite crystal variables were compared, using an in vitro crystal growth system and 24-hour samples of pooled urine obtained from 4 cats. Urine samples were placed in wells of cell culture plates, increasing concentrations of ammonium hydroxide were added to adjacent wells to stimulate struvite crystal growth, and the plates were incubated at 37 C. The effect of albumin and THP on crystal growth in sample wells was compared with that in control wells, without protein addition, in the same plate. Crystal growth was assessed by determination of number of crystals and supersaturation index, a scale of crystal habit at different degrees of supersaturation, by use of direct visualization with an inverted microscope. Albumin addition did not have significant effect on either crystal number or supersaturation, compared with controls. Addition of THP significantly (P < 0.05) increased crystal number and supersaturation index. It was concluded that THP significantly (P < 0.05) promoted growth of struvite crystals in feline urine, and thus, may have a role in feline struvite uroliths and struvite urethral plug formation.

Ammonia↗
PubMed1994

Cytosolic Ca2+ concentration determinations in neutrophils stimulated by monosodium urate and calcium pyrophosphate crystals: effect of protein adsorption.

OBJECTIVE: To develop methods to correct for light scattering artifacts produced by monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) crystals in the measurement of the levels of cytosolic free calcium, [Ca2+]i, in neutrophils and to quantitate the extent of association of neutrophils with crystals. METHODS: The association of neutrophils with crystals through binding of crystals onto the neutrophil plasma membrane and/or internalization was quantitated by mixing crystals with diphenyl hexatriene (DPH) loaded neutrophils and monitoring the fluorescence intensity of supernatants ("free" neutrophils) following gravity sedimentation of "free" crystals and neutrophils with bound/internalized crystals. The effects of precoating CPPD and MSUM crystals with IgG, plasma, serum and BSA on crystal-neutrophil association and [Ca2+]i were determined. RESULTS: MSUM produced a large increase in [Ca2+]i that reached maximal values in less than 1 min, whereas CPPD produced a smaller and slower increase in [Ca2+]i. IgG adsorbed onto CPPD increased the rate and extent of increase in [Ca2+]i. Heparinized plasma and serum adsorbed onto CPPD increased the extent but not the rate of increase in [Ca2+]i. CONCLUSION: [Ca2+]i increases induced by MSUM were not affected by IgG, serum or heparinized plasma precoating. IgG, serum, or heparinized plasma adsorbed into CPPD amplified neutrophil activation increasing the extent of the increase in [Ca2+]. Neither MSUM nor CPPD induced increases in [Ca2+]i were sensitive to precoating crystals with bovine serum albumin.

Adsorption↗
PubMed1996

Regulation of alkaline phosphatase: implications for calcium pyrophosphate dihydrate crystal dissolution and other alkaline phosphatase functions.

OBJECTIVE: Alkaline phosphatase (ALP), an enzyme with pyrophosphatase (PPiase) activity can dissolve calcium pyrophosphate dihydrate (CPPD) crystals. We studied the effects of enzyme inhibitors such as bisphosphonates, orthovanadate, calcium, cadmium, and ascorbic acid on PPiase activity of ALP as well as on phosphate ester hydrolysis (Pase) activity and compared these effects to those on CPPD crystal dissolution. METHOD: An in vitro model system for crystal enzyme interaction was used to assess CPPD crystal dissolution. RESULTS: Bisphosphonates inhibited ALP Pase activity more than ALP PPiase activity at the same concentrations. Calcium inhibited ALP PPiase activity, but not ALP Pase activity. Orthovanadate and cadmium inhibited ALP PPiase activity more than ALP Pase at the same concentrations. The inhibition rates of ALP PPiase at the same concentrations were orthovanadate > cadmium > calcium. Although ALP Pase activity was not inhibited, at high concentrations, ascorbic acid slightly inhibited ALP PPiase activity. Bisphosphonates at high concentrations inhibited ALP CPPD crystal dissolution. The strong inhibitory effects of bisphosphonates on ALP CPPD crystal dissolution compared to those on ALP PPiase activity suggest that bisphosphonates inhibit crystal dissolution by their affinity for the CPPD crystal surface. Calcium, orthovanadate, and cadmium inhibited ALP CPPD dissolution. The inhibition rates of ALP CPPD dissolution at the same concentrations were cadmium > calcium > orthovanadate. Ascorbic acid at high concentrations enhanced ALP CPPD dissolution. CONCLUSION: These effects of different inhibitors on ALP PPiase and CPPD dissolution suggest that ALP CPPD crystal dissolution depends on binding of ALP CPPD crystals as well as the PPiase activity of the bound ALP. Because of its ubiquitous and broad phosphatase activity including PPiase activity, ALP may have a critical role in cell energy metabolism.

Alkaline Phosphatase↗
PubMed1976

Crystallization of proteins from polyethylene glycol.

Twenty-two proteins available to this laboratory were examined for their ability to crystallize from polyethylene glycol (PEG). Of the 22, 8 had been crystallized previously by conventional means and the remainder had yielded no crystals by other methods. Using a single broad screen with four different concentrations of five different molecular weight sizes of polyethylene glycol, 13 of these proteins were crystallized. Of the 13, 6 proteins were crystallized for the first time, one yielded crystals readily where only infrequent and irreproducible results were obtained before, and one other produced excellent quality crystals where only very disordered or twinned crystals were previously grown. In only one case did polyethylene glycol fail to yield crystals where other agents had succeeded. It is concluded that polyethylene glycol may be the best initial trial reagent for crystallization of proteins for x-ray diffraction analyses.

Animals↗
PubMed1987

Incorporation of strontium into the calcium carbonate crystals of the endolymphatic sac in the tree frog (Hyla arborea japonica).

Tree frogs were loaded with strontium chloride (SrCl2). The incorporation of strontium metal into the calcium carbonate (CaCO3) crystals located both in the inner ear and in the endolymphatic sac was studied by x-ray microanalysis (XMA) and scanning electron microscopy (SEM). In the inner ear, strontium was not recognized except for traces in a few crystals. When observed by SEM, these crystals had a faceted body and two pointed ends with rather smooth surfaces. However, in the endolymphatic sac, which greatly expands into the spinal canal, strontium was clearly present at every surface of all crystals. Careful examinations by point and line XMA revealed that strontium x-ray counts were highest at the pointed ends and decreased sharply and then gradually toward the equator of the crystals. SEM observations revealed that the crystals in the endolymphatic sac always had rough and irregular surfaces regardless of their shapes and sizes. Calcium was always found in crystals of both organs. Except for calcium and strontium, other elements including sodium and heavier elements were negligible in XMA. These findings suggest that strontium is incorporated into the crystals only in the endolymphatic sac, and the rough-surfaced covering of these crystals reflects newly deposited strontium salt. It seems to indicate that these crystals grow predominantly by accretion.

Animals↗
PubMed2005

Innate immunity conferred by Toll-like receptors 2 and 4 and myeloid differentiation factor 88 expression is pivotal to monosodium urate monohydrate crystal-induced inflammation.

OBJECTIVE: In gout, incompletely defined molecular factors alter recognition of dormant articular and bursal monosodium urate monohydrate (MSU) crystal deposits, thereby inducing self-limiting bouts of characteristically severe neutrophilic inflammation. To define primary determinants of cellular recognition, uptake, and inflammatory responses to MSU crystals, we conducted a study to test the role of Toll-like receptor 2 (TLR-2), TLR-4, and the cytosolic TLR adapter protein myeloid differentiation factor 88 (MyD88), which are centrally involved in innate immune recognition of microbial pathogens. METHODS: We isolated bone marrow-derived macrophages (BMDMs) in TLR-2-/-, TLR-4-/-, MyD88-/-, and congenic wild-type mice, and assessed phagocytosis and cytokine expression in response to endotoxin-free MSU crystals under serum-free conditions. MSU crystals also were injected into mouse synovium-like subcutaneous air pouches. RESULTS: TLR-2-/-, TLR-4-/-, and MyD88-/- BMDMs demonstrated impaired uptake of MSU crystals in vitro. MSU crystal-induced production of interleukin-1beta (IL-1beta), tumor necrosis factor alpha, keratinocyte-derived cytokine/growth-related oncogene alpha, and transforming growth factor beta1 also were significantly suppressed in TLR-2-/- and TLR-4-/- BMDMs and were blunted in MyD88-/- BMDMs in vitro. Neutrophil influx and local induction of IL-1beta in subcutaneous air pouches were suppressed 6 hours after injection of MSU crystals in TLR-2-/- and TLR-4-/- mice and were attenuated in MyD88-/- mice. CONCLUSION: The murine host requires TLR-2, TLR-4, and MyD88 for macrophage activation and development of full-blown neutrophilic, air pouch inflammation in response to MSU crystals. Our findings implicate innate immune cellular recognition of naked MSU crystals by specific TLRs as a major factor in determining the inflammatory potential of MSU crystal deposits and the course of gouty arthritis.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Photonic crystals--a step towards integrated circuits for photonics.

The field of photonic crystals has, over the past few years, received dramatically increased attention. Photonic crystals are artificially engineered structures that exhibit a periodic variation in one, two, or three dimensions of the dielectric constant, with a period of the order of the pertinent light wavelength. Such structures in three dimensions should exhibit properties similar to solid-state electronic crystals, such as bandgaps, in other words wavelength regions where light cannot propagate in any direction. By introducing defects into the periodic arrangement, the photonic crystals exhibit properties analogous to those of solid-state crystals. The basic feature of a photonic bandgap was indeed experimentally demonstrated in the beginning of the 1990s, and sparked a large interest in, and in many ways revitalized, photonics research. There are several reasons for this attention. One is that photonic crystals, in their own right, offer a proliferation of challenging research tasks, involving a multitude of disciplines, such as electromagnetic theory, nanofabrication, semi-conductor technology, materials science, biotechnology, to name a few. Another reason is given by the somewhat more down-to-earth expectations that photonics crystals will create unique opportunities for novel devices and applications, and contribute to solving some of the issues that have plagued photonics such as large physical sizes, comparatively low functionality, and high costs. Herein, we will treat some basics of photonic crystal structures and discuss the state-of-the-art in fabrication as well give some examples of devices with unique properties, due to the use of photonic crystals. We will also point out some of the problems that still remain to be solved, and give a view on where photonic crystals currently stand.

Journal Article↗
PubMed1990

High resolution SE-I SEM study of enamel crystal morphology.

Until recently high resolution TEM was the only imaging mode capable of probing the atomic lattice structure of crystals composing tooth enamel. Studies designed to determine the polyhedral shape of normal enamel crystals and initiation of carious lesions in enamel crystals were hampered and limited by interpretation of two-dimensional TEM images from thin section and freeze fracture replica specimens lacking depth of field. The newly developed SE-I signal mode for SEM (SE-I/SE-II ratio) can produce images of enamel crystals approaching beam diameter dimensions (0.7-2.0 nm), rivaling the resolution of the TEM technique and generating topographic contrasts for three dimensional imaging at very high magnification (approximately 1,000,000X). Ultrathin chromium (Cr) films generate enriched high resolution SE-I contrasts of enamel crystal surfaces and when imaged using an immersion lens field emission SEM operated at high voltage (20-30 KeV) produce unsurpassed topographic contrasts. Since the grain size of Cr is below the resolution of any SEM and is ultrathin (approximately 1 nm), then SE-I images can provide a more accurate representation of enamel crystal structure than TEM methodologies. Our SE-I SEM observations of normal human enamel crystals reveal fractured spicules which contain angled flat surfaces delineated by a prominent 2 nm wide SE-I edge brightness contrast. Although microscopic observations often show crystals which are hexagonal in cross-section, in both SEM and TEM many other growth habits, including rectangular or irregular crystals (30-40 nm in width) which contain "notches," are also observed. More detailed morphological studies are therefore required to determine the most likely habit planes and their relevance to the function of the enamel crystals.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromium↗
PubMedDate not supplied

Irregular snow crystals: structural features as revealed by low temperature scanning electron microscopy.

For nearly 50 years, investigators using light microscopy have vaguely alluded to a unique type of snow crystal that has become known as an irregular snow crystal. However, the limited resolution and depth-of-field of the light microscope has prevented investigators from characterizing these crystals. In this study, a field-emission scanning electron microscope, equipped with a cold stage, was used to document the structural features, physical associations, and atmospheric metamorphosis of irregular snow crystals. The crystals appear as irregular hexagons, measuring 60 to 90 mm across, when viewed from the a-axis. Their length (c-axis) rarely exceeds the diameter. The irregular crystals are occasionally found as secondary particles on other larger forms of snow crystals; however, they most frequently occur in aggregates consisting of more than 100 irregular crystals. In the aggregates, the irregular crystals have their axes oriented parallel to one another and, collectively, tend to form columnar structures. Occasionally, these columnar structures exhibit rounded faces along one side, suggesting atmospheric metamorphoses during formation and descent. In extreme cases of metamorphoses, the aggregates would be difficult to distinguish from graupel. Frost, consisting of irregular crystals, has also been encountered, suggesting that atmospheric conditions that favor their growth can also occur terrestrially.

Journal Article↗
PubMed1995

Preliminary cryocrystallographic study of the mitochondrial cytochrome bc1 complex: improved crystallization and flash-cooling of a large membrane protein.

Ubiquinol-cytochrome c reductase is a crucial integral membrane protein in the mitochondrial respiratory cycle. Eleven subunits containing three cytochrome heme groups and a 2Fe-2S Rieske center make up this 240 kDa enzyme complex. Previously, many different crystal forms of the bc1 complex have displayed diffraction to as far as 4.5 A. However, rapid degradation of the protein in the X-ray beam at room temperature has obstructed the collection of a full data set from a single crystal. As slight heterogeneities between crystals severely hampered merging of data from different crystals, we sought a method to stabilize the protein crystal in the X-ray beam in order to collect a full data set from one crystal sample. To this end, water soluble protein crystals are frequently flash-cooled to cryogenic temperatures; however, there is no report of cryocrystallography for membrane proteins. In this communication, we report on a successful experiment in which flash-cooled bc1 membrane protein crystals have given rise to sustained diffraction over a 60 hour data collection period at a synchrotron source. Furthermore, we present an improved purification and crystallization protocol yielding crystals readily diffracting out to 3.3 A. These results should greatly aid in the future realization of the molecular structure of the bc1 complex as well as other membrane proteins.

Animals↗
PubMed1994

Analysis of electrostatic interactions in ribonuclease A monoclinic crystal.

The electrostatic field in ribonuclease A monoclinic crystal is computed. A calculation of the electrostatic free energy of interaction between one molecule and its neighbours in the crystal lattice is performed in three crystal planes. It is shown that at the pH value of crystallization for the polymorphic form under investigation the total electrostatic free energy in the monoclinic crystal of ribonuclease A already crystallized is equal to zero. However, the electrostatic free energy along the crystal axis has different values. Electrostatic forces play a negative role for the crystal stability along the alpha-axis while along the other axis the electrostatic interaction supports the crystal state. The electrostatic potential distribution of a ribonuclease A monoclinic crystal at pH 6 is plotted and it is demonstrated that these interactions have a different character in each crystallographic plane.

Crystallography↗
PubMed2006

Thermodynamic and kinetic aspects of fat crystallization.

Naturally occurring fats are multi-component mixtures of triacylglycerols (TAGs), which are triesters of fatty acids with glycerol, and of which there are many chemically distinct compounds. Due to the importance of fats to the food and consumer products industries, fat crystallization has been studied for many years and many intricate features of TAG interactions, complicated by polymorphism, have been identified. The melting and crystallization properties of triacylglycerols are very sensitive to even small differences in fatty acid composition and position within the TAG molecule which cause steric hindrance. Differences of fatty acid chain length within a TAG lead to packing imperfections, and differences in chain lengths between different TAG molecules lead to a loss of intersolubility in the solid phase. The degree of saturation is hugely important as the presence of a double bond in a fatty acid chain causes rigid kinks in the fatty acid chains that produce huge disruption to packing structures with the result that TAGs containing double bonds have much lower melting points than completely saturated TAGs. All of these effects are more pronounced in the most stable polymorphic forms, which require the most efficient molecular packing. The crystallization of fats is complicated not just by polymorphism, but also because it usually occurs from a multi-component melt rather than from a solvent which is more common in other industrial crystallizations. This renders the conventional treatment of crystallization as a result of supersaturation somewhat meaningless. Most studies in the literature consequently quantify crystallization driving forces using the concept of supercooling below a distinct melting point. However whilst this is theoretically valid for a single component system, it can only at best represent a rough approximation for natural fat systems, which display a range of melting points. This paper reviews the latest attempts to describe the sometimes complex phase equilibria of fats using fundamental relationships for chemical potential that have so far been applied to individual species in melts of unary, binary and ternary systems. These can then be used to provide a framework for quantifying the true crystallization driving forces of individual components within a multi-component melt. These are directly related to nucleation and growth rates, and are also important in the prediction of polymorphic occurrence, crystal morphology and surface roughness. The methods currently used to evaluate induction time, nucleation rate and overall crystallization rate data are also briefly described. However, mechanistic explanations for much of the observed crystallization behaviour of TAG mixtures remain unresolved.

Journal Article↗