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PubMed1994

Renal epithelial cells rapidly bind and internalize calcium oxalate monohydrate crystals.

Renal tubular fluid is supersaturated with calcium and oxalate ions, which can nucleate to form crystals of calcium oxalate monohydrate (COM), the most abundant constituent of kidney stones. However, the mechanisms by which nascent crystals are retained in the nephron and then grow into kidney stones are unclear. An interaction of COM crystals with the surface of renal epithelial cells could be a critical initiating event in nephrolithiasis. To investigate this possibility we used cultures of monkey kidney epithelial cells (BSC-1 line) as a model system and found that [14C]COM crystals bound to the cell surface within seconds. Scanning electron microscopy revealed that crystals bind first to apical microvilli, which subsequently migrate over the crystalline surface. When visualized by transmission electron microscopy, intracellular crystals were located within vesicles. Cytoskeletal responses to crystal uptake were sought by immunofluorescence microscopy, which revealed concentration of F-actin at sites of crystal contact as well as a generalized reorganization of the intermediate filament network containing cytokeratin 8. Uptake of COM crystals did not adversely affect renal epithelial cell growth, and internalized crystals were apparently distributed to daughter cells during division. Rapid adherence of COM crystals to the apical surface of tubular epithelial cells could promote crystal retention in the kidney. Elucidation of factors that regulate this process may provide insight into the pathogenesis of nephrolithiasis.

Animals↗
PubMed1973

Octahedral crystals in Phycomyces. II.

"Phycomyces blakesleeanus" sporangiophores contain octahedral crystals throughout their cytoplasm and vacuole. More octahedral crystals were found in the wild-type strain G5 (+) than in the beta-carotene-deficient mutant C5 (-), and much more than in the mutant C141 (-), which is sensitive to only high light intensity. In the wild type, the number of crystals per sporangiophore increased until the sporangiophore reached stage IV, and then decreased. Stage I contained the most crystals per unit volume. Cultures grown in darkness had the maximum number of crystals. Under high light intensity, there was an overall reduction of crystals. The crystals are regular octahedrons. The crystals were isolated from the sporangiophores by a method of sucrose density-gradient centrifugation. They contain nearly 95% protein, are stable in organic solvents, but can be solubilized in buffer solution above pH 9.5 and below 2.5. The crystals weakly fluoresce with an emission peak at 540 nm, which is affected by irradiation with white light. Absorption spectra of freshly prepared crystals show absorption maxima around 265-285 nm, 350-380 nm, and 450-470 nm. These absorption peaks for the crystals are close to those of the phototropic and light-growth action spectra. These data suggest that the crystals may contain a flavoprotein which may be the photoreceptor pigment of "Phycomyces".

Carotenoids↗
PubMed2002

Crystal-cell interaction in the pathogenesis of kidney stone disease.

PURPOSE OF REVIEW: Renal stone formation depends not so much on the formation of crystals, but on their retention in the kidney. Evidence has emerged that crystal retention is caused predominantly by the adherence of crystals to the epithelial cells lining the renal tubules. Understanding the mechanisms involved in crystal retention could lead to new therapeutic approaches for interfering with the renal stone-forming process in patients. Cell-culture studies have been performed to obtain insights into the susceptibility of the cell surface to crystal attachment, and to uncover cell-surface crystal-binding molecules. This review aims to put the relevant publications of the last decade in perspective. RECENT FINDINGS: Crystal-cell interaction has been investigated by using various renal tubular cell types in culture. Such studies have yielded several candidate crystal-binding molecules, including phosphatidylserine, sialic acid, collagen IV, osteopontin and, recently, hyaluronan. SUMMARY: Here, the results obtained in crystal-binding studies are recapitulated, compared and evaluated. Arguments are provided in support of the view that many of the proposed crystal-binding molecules could be linked in the series of events resulting in crystal retention. Under pathological conditions, pericellular matrices rich in the polysaccharide hyaluronan are proposed as the key binding substance for crystals at the surface of renal tubular cells.

Animals↗
PubMed2003

Isolation of a crystal matrix protein associated with calcium oxalate precipitation in vacuoles of specialized cells.

The formation of calcium (Ca) oxalate crystals is considered to be a high-capacity mechanism for regulating Ca in many plants. Ca oxalate precipitation is not a stochastic process, suggesting the involvement of specific biochemical and cellular mechanisms. Microautoradiography of water lettuce (Pistia stratiotes) tissue exposed to 3H-glutamate showed incorporation into developing crystals, indicating potential acidic proteins associated with the crystals. Dissolution of crystals leaves behind a crystal-shaped matrix "ghost" that is capable of precipitation of Ca oxalate in the original crystal morphology. To assess whether this matrix has a protein component, purified crystals were isolated and analyzed for internal protein. Polyacrylamide gel electrophoresis revealed the presence of one major polypeptide of about 55 kD and two minor species of 60 and 63 kD. Amino acid analysis indicates the matrix protein is relatively high in acidic amino acids, a feature consistent with its solubility in formic acid but not at neutral pH. 45Ca-binding assays demonstrated the matrix protein has a strong affinity for Ca. Immunocytochemical localization using antibody raised to the isolated protein showed that the matrix protein is specific to crystal-forming cells. Within the vacuole, the surface and internal structures of two morphologically distinct Ca oxalate crystals, raphide and druse, were labeled by the antimatrix protein serum, as were the surfaces of isolated crystals. These results demonstrate that a specific Ca-binding protein exists as an integral component of Ca oxalate crystals, which holds important implications with respect to regulation of crystal formation.

Amino Acids↗
PubMed2004

Crystallization and preliminary crystallographic analysis of endonuclease VIII in its uncomplexed form.

The Escherichia coli DNA repair enzyme endonuclease VIII (EndoVIII or Nei) excises oxidized pyrimidines from damaged DNA substrates. It overlaps in substrate specificity with endonuclease III and may serve as a back-up for this enzyme in E. coli. The three-dimensional structure of Nei covalently complexed with DNA has been recently determined, revealing the critical amino-acid residues required for DNA binding and catalytic activity. Based on this information, several site-specific mutants of the enzyme have been tested for activity against various substrates. Although the crystal structure of the DNA-bound enzyme has been fully determined, the important structure of the free enzyme has not previously been analyzed. In this report, the crystallization and preliminary crystallographic characterization of DNA-free Nei are described. Four different crystal habits are reported for wild-type Nei and two of its catalytic mutants. Despite being crystallized under different conditions, all habits belong to the same crystal form, with the same space group (I222) and a similar crystallographic unit cell (average parameters a = 57.7, b = 80.2, c = 169.7 A). Two of these crystal habits, I and IV, appear to be suitable for full crystallographic analysis. Crystal habit I was obtained by vapour diffusion using PEG 8000, glycerol and calcium acetate. Crystal habit IV was obtained by a similar method using PEG 400 and magnesium chloride. Both crystals are mechanically strong and stable in the X-ray beam once frozen under cold nitrogen gas. A full diffraction data set has recently been collected from a wild-type Nei crystal of habit I (2.6 A resolution, 85.2% completeness, Rmerge = 9.8%). Additional diffraction data were collected from an Nei-R252A crystal of habit IV (2.05 A resolution, 99.9% completeness, Rmerge = 6.0%) and an Nei-E2A crystal of habit IV (2.25 A resolution, 91.7% completeness, Rmerge = 6.2%). These diffraction data were collected at 95-100 K using a synchrotron X-ray source and a CCD area detector. All three data sets are currently being used to obtain crystallographic phasing via molecular-replacement techniques.

Crystallization↗
PubMed2006

Structural phase transition of monoclinic crystals of hen egg-white lysozyme.

Two monoclinic crystals (space group P2(1)) of hen egg-white lysozyme, a type I crystal grown at room temperature in a D2O solution with pD 4.5 containing 2%(w/v) sodium nitrate and a type II crystal grown at 313 K in a 10%(w/v) sodium chloride solution with pH 7.6, were each transformed into another monoclinic crystal with the same space group by dehydration-induced phase transition. Changes in X-ray diffraction were recorded to monitor the progress of the crystal transformation, which started with the appearance of diffuse streaks. In both crystals, the intensity of h + l odd reflections gradually weakened and finally disappeared on completion of the transformation. X-ray diffraction in the intermediate state indicated the presence of lattices of both the native and transformed crystals. In the native type I crystal, two alternate conformations were observed in the main chain of the region Gly71-Asn74. One conformer bound a sodium ion which was replaced with a water molecule in the other conformer. In the transformed crystal, the sodium ion was removed and the main-chain conformation of this region was converted to that of the water-bound form. The transformed crystal diffracted to a higher resolution than the native crystal, while the peak width of the diffraction spots increased. Analysis of the thermal motion of protein molecules using the TLS model has shown that the enhancement of the diffraction power in the transformed crystal is mainly ascribable to the suppression of rigid-body motion owing to an increase in intermolecular contacts as a result of the loss of bulk solvent.

Animals↗
PubMed1999

A general method for co-crystallization of concanavalin A with carbohydrates.

A small grid of conditions has been developed for co-crystallization of the plant lectin concanavalin A (conA) and polysaccharides. Crystals have been obtained of complexes of conA with alpha1-2 mannobiose, 1-methyl alpha1-2 mannobiose, fructose, a trisaccharide and a pentasaccharide. The crystals diffract to resolutions of 1. 75-2.7 A using a copper rotating-anode source. The crystals are grown in the presence of polyethylene glycol 6K [10-20%(w/v)] at around pH 6.0. Optimization for each particular carbohydrate requires small adjustments in the conditions; however, all complexes give some crystalline precipitate in this limited grid. The alpha1-2 mannobiose complex crystals diffract to 1.75 A with space group I222 and cell dimensions a = 91.7, b = 86.8, c = 66.6 A. One monomer is present in the asymmetric unit. The 1-methyl alpha1-2 mannobioside complex crystallizes in space group P212121, cell dimensions a = 119. 7, b = 119.7, c = 68.9 A and diffract to 2.75 A. One tetramer is present in the asymmetric unit. Two crystal forms of the conA-fructose complex have been obtained. The first has space group P212121, cell dimensions a = 121.7, b = 119.9, c = 67.3 A with a tetramer in the asymmetric unit and diffracts to 2.6 A. The second crystallizes in space group C2221, cell dimensions a = 103.3, b = 117.9, c = 254.3 A with two dimers in the asymmetric unit and diffracts to 2.42 A. Structures and crystallization of the trisaccharide-conA and pentasaccharide-conA complexes have already been reported. In all complexes, the protein is found as a tetramer, although varying combinations of non-crystallographic and crystallographic symmetry are involved in generating the tetramer. The precise packing of the tetramer varies from crystal to crystal and it is likely that this variability facilitates crystallization.

Carbohydrate Sequence↗
PubMed1999

Crystallization and preliminary X-ray analysis of alpha-D-glucuronidase from Bacillus stearothermophilus T-6.

alpha-D-Glucuronidases cleave the alpha-1,2-glycosidic bond of the 4-O-methyl-alpha-D-glucuronic acid side chain in xylan. Of the xylan-debranching hydrolases, these enzymes are the least studied and characterized. The alpha-glucuronidase gene (aguA) from Bacillus stearothermophilus T-6 has been cloned, sequenced and overproduced in Escherichia coli. The gene encodes for a protein of 679 amino acids with a calculated molecular weight of 78480 and a pI of 5.42. alpha-Glucuronidase T-6 shows high homology to the alpha-glucuronidases of Thermotoga maritima (60% identity) and of Tri-choderma reesei (44% identity). Based on the amino-acid sequence similarity, it is likely that these enzymes represent a new class of glycosyl hydrolases. Crystallographic studies of alpha-glucuronidase T-6 were initiated to study the mechanism of catalysis, as well as to provide a structural basis for rational introduction of enhanced thermostability by site-specific mutagenesis. In this report, the crystallization and preliminary crystallographic characterization of the native alpha-glucuronidase T-6 enzyme is described. Two crystal forms were found suitable for detailed crystal structure analysis. The T1 form was obtained by the vapour-diffusion method using PEG 4000 as a precipitant and 2-propanol as an organic additive. The crystals belong to a primitive tetragonal crystal system (space group P41212 or P43212) with unit-cell dimensions a = b = 76.1 and c = 331.2 A. These crystals are mechanically strong, are stable in the X--ray beam and diffract X-rays to better than 2.4 A resolution. A full 3.0 A resolution diffraction data set (97.3% completeness, Rmerge 9.8%) has recently been collected on one crystal at room temperature using a rotating-anode X-ray source and an R-AXIS IIc imaging-plate detector. The M1 form was obtained and characterized by similar techniques. The best crystallization occurred at a slightly lower pH and a lower concentration of 2-propanol. The crystals belong to a primitive monoclinic crystal system (space group P21) with unit-cell dimensions a = 65.8, b = 127.4, c = 96.6 A and beta = 97.9 degrees. These crystals are also quite strong and stable, and diffract to better than 2.8 A resolution. A full 2.8 A resolution diffraction data set (96.2% completeness, Rmerge 7.6%) has recently been collected on one crystal at room temperature using the same R-AXIS IIc setup. Both forms are currently being used to obtain crystallographic phasing via isomorphous heavy-atom derivatives and selenomethionine MAD experiments.

Bacterial Proteins↗
PubMed2000

Lactate dehydrogenase from the hyperthermophilic archaeon Methanococcus jannaschii: overexpression, crystallization and preliminary X-ray analysis.

L(+)-Lactate dehydrogenase (LDH) is a key enzyme in anaerobic metabolism which converts pyruvate to lactate. LDH from the hyperthermophilic archaebacterium Methanococcus jannaschii has been overexpressed in Escherichia coli and crystallized in two crystal forms at 297 K using 2-methyl-2,4-pentanediol as precipitant. Type I crystals grew rapidly and diffracted to at least 2.8 A Bragg spacing upon exposure to Cu Kalpha X-rays. X-ray diffraction data to 2.9 A have been collected from a native crystal. The type I crystal is tetragonal, belonging to the space group P4(2)2(1)2, with unit-cell parameters a = b = 99.74, c = 170.00 A. The asymmetric unit contains two LDH subunits, with a corresponding crystal volume per protein mass (V(m)) of 3.05 A(3) Da(-1) and a solvent content of 59.7%. Type II crystals, which grew more slowly, diffracted to at least 1.8 A Bragg spacing upon exposure to Cu Kalpha X-rays. X-ray diffraction data to 1.9 A have been collected from a native crystal. The type II crystal is orthorhombic, belonging to the space group P2(1)2(1)2, with unit-cell parameters a = 47.65, b = 125.10, c = 58.08 A. The asymmetric unit contains a single LDH subunit, with a corresponding crystal volume per protein mass (V(m)) of 2.50 A(3) Da(-1) and a solvent content of 50.8%. Therefore, the type II crystal is more suitable for high-resolution structure determination than the type I crystal.

Cloning, Molecular↗
PubMed1997

Does urinary oxalate interfere with the inhibitory role of glycosaminoglycans and semisynthetic sulfated polysaccharides in calcium oxalate crystallization?

OBJECTIVES: Previously it was shown that the polysaccharide G872 in vitro strongly inhibits calcium oxalate monohydrate crystallization processes. However, when rats on a stone-inducing diet of ethylene glycol plus vitamin D3 are given this polysaccharide, no changes in the urine capacity for crystallization inhibition were found. We investigated here how the inhibitory action of polysaccharides changes under high oxalate conditions, as they exist in the stone inducing diet. METHODS: Calcium oxalate monohydrate (COM) crystals were incubated in a series of 0.05 M PBS buffers containing polysaccharides with increasing oxalate concentrations (0-0.4 mmol/l). The coated crystals were collected, washed and resuspended in an artificial urine. We then measured the zeta potential of the crystals, using a Coulter DELSA 440, and the initial rates for crystal growth and agglomeration, using the Coulter Multisizer II. RESULTS: Addition of oxalate to the medium shifts the negative zeta potential distribution of COM crystals coated by polysaccharides in positive direction. Particle size analysis demonstrated that the initial rates of COM crystal growth and agglomeration responding to oxalate concentration changes (0.1-->0.4 mmol/l) in the presence of G872 (0.2 mg/l) are approximately 2.5 times faster than that in the absence of G872. CONCLUSIONS: Oxalate interferes with the binding of polysaccharides to crystals. This can be envisioned to occur through changes in the crystal surface properties or by induction of functional and secondary structural changes of urinary macromolecular inhibitors such as GAGs, resulting in a decrease of their inhibitory activity against COM crystallization. Thus, in urine, a high oxalate may increase the rate of crystallization both by increasing the supersaturation and by decreasing the inhibitory potential of the urine.

Calcium Oxalate↗
PubMed1995

Crystal bonding--an adhesive system with a future?

The primary objective of this investigation was to evaluate the crystal bonding technique and to: (1) study the effect on crystal size, coverage, and morphology of altering the concentration of lithium sulphate in a crystal growth solution; (2) investigate the potential for reattachment of brackets, following the use of the crystal growth technique; (3) assess the deterioration of the crystal growth conditioning solution with time; (4) investigate the influence of polyacrylic acid enamel pretreatment on the bond strength subsequently achieved with the crystal growth technique; and (5) confirm that the use of crystal growth solutions to attach orthodontic brackets, results in minimal enamel surface change on debond when compared to the acid etch technique. The results of the study suggest that altering the lithium sulphate concentration of the crystal growth solution results in little alteration in the size, morphology, and coverage of crystal produced. The crystal growth technique allows reattachment of orthodontic brackets with no obvious deterioration in bond strength and the crystal growth solution shows little deterioration in performance after storage for 2 years. However, the hope that the use of the crystal growth technique might be a routine and valid alternative to the acid etch technique could not be confirmed.

Acid Etching, Dental↗
PubMed2004

Numerical analysis of the depletion zone formation around a growing protein crystal.

It is expected that a protein depletion zone and an impurity depletion zone are formed around a crystal during protein crystal growth if the diffusion field around the crystal is not disturbed. The growth rate of the crystal may be decreased and the impurity uptake may be suppressed to result in highly ordered crystals if these zones are not disturbed. It is well known that a microgravity environment can reduce convective fluid motion, and this is thought to disturb the depletion zones. Therefore, we expect that crystals grown in space can attain better quality than those grown on the ground. In this study, we estimate the depletion zone formation numerically and discuss the results of crystallization in space experiments. In case of alpha-amylase, most of the crystals form a cluster-like morphology on the ground using PEG 8000 as a precipitant. However, in space, we have obtained a single and high-quality crystal grown from the same sample compositions. We have measured the viscosity of the solution, the diffusion coefficient, and the growth rate of protein crystals on the ground. Applying numerical analysis to these values a significant depletion zone was expected to form mainly due to higher values of the viscosity. This might be one of the main reasons for better quality single crystals grown in space, where the depletion zone is thought to remain undisturbed. For protein crystallization experiments, salts are widely used as a precipitant. However, in that case, reduced concentration depletion zone effects can be expected because of a low viscosity. Therefore, if it is possible to increase the viscosity of the protein solution by means of an additive, the depletion zone formation effect would be enhanced to provide a technique that would be especially effective in space.

Aspergillus oryzae↗
PubMed2004

Scientific approach to the optimization of protein crystallization conditions for microgravity experiments.

The National Space Development Agency of Japan (NASDA) developed a practical protocol to optimize protein crystallization conditions for microgravity experiments. This protocol focuses on the vapor diffusion method using high density protein crystal growth (HDPCG)--hardware developed by the University of Alabama, Birmingham--that flew on the STS-107 mission. The objective of this development was to increase the success rate of microgravity experiments by setting crystallization conditions based on knowledge of crystal growth and fluid dynamics. The protocol consists of four steps: (1) phase diagram preparation, (2) estimation of condensation rate in the vapor diffusion method, (3) fluid dynamic property measurement, and (4) fluid dynamic simulation. First, a phase diagram was constructed. Crystallization characteristics were investigated by a microbatch method. The data were recalculated based on classical nucleation theory and the crystallization boundary was determined as a function of time. The second step was to develop a practical model to estimate the condensation rate of the crystallizing solution, including protein and precipitant, as a function of the precipitant concentration and solution volume. By considering the crystallization map and the vapor diffusion condensation model we were able to optimize the crystallization conditions that generate crystals in the desired time. This was particularly important in a shuttle mission whose mission duration is limited. The third step was fluid dynamic property measurement necessary for fluid dynamics simulation and crystal growth study. The last step was to estimate the mass transport in space on the basis of the fluid dynamics simulation transport model. It turned out that neither the vapor phase nor the solution phase was seriously affected by gravity until nucleation provided the hardware was set in a normal direction. Therefore, we concluded that the optimized crystallization conditions could be directly applied to microgravity experiments. By completing the approach, we were able to control the time for nucleation in the vapor diffusion method.

Animals↗
PubMed2003

Effect of crystallization behavior of polyethylene glycol 6000 on the properties of granules prepared by fluidized hot-melt granulation (FHMG).

The objective of this study was to investigate the effect of the crystallization behavior of Macrogol 6000 (polyethylene glycol 6000; PEG 6000), used as a binder, during the solidification process on the properties of mononucleic granules prepared by the fluidized hot-melt granulation (FHMG) technique. Crystallization of PEG 6000 from molten liquid was investigated using differential scanning calorimetry (DSC) and hot stage microscopy. The results obtained from the measurement of isothermal crystallization demonstrated that crystallization of PEG 6000 was either slow or rapid. Analysis based on solid-state decomposition showed that slow crystallization was due to the two-dimensional growth of nuclei mechanism, while rapid crystallization was due to the three-dimensional growth of nuclei mechanism. Observation of the crystallization of PEG 6000 by hot stage microscopy supported the existence of two different crystallization mechanisms. Granules containing PEG 6000 that underwent rapid crystallization during FHMG showed a significantly higher fraction powder under 150 microm in diameter. This was caused by the loss of powder particles from the surface of mononucleic granules during the solidification process, because many cracks were observed after crystallization of PEG 6000 with a short isothermal crystallization time (ICT) due to the reduced of sticking of particles. The results of this study suggested that the crystallization behavior of the binder during the solidification process of FHMG can influence the properties of the resultant granules, such as particle size distribution, content uniformity or taste masking. It was also indicated that measuring the ICT using DSC was a useful method to classify PEG 6000.

Calorimetry, Differential Scanning↗
PubMed2007

Annealing macromolecular crystals.

The process of crystal annealing has been used to improve the quality of diffraction from crystals that would otherwise be discarded for displaying unsatisfactory diffraction after flash cooling. Although techniques and protocols vary, macromolecular crystals are annealed by warming the flash-cooled crystal, then flash cooling it again. To apply macromolecular crystal annealing, a flash-cooled crystal displaying unacceptably high mosaicity or diffraction from ice is removed from the goniometer and immediately placed in cryoprotectant buffer. The crystal is incubated in the buffer at either room temperature or the temperature at which the crystal was grown. After about 3 min, the crystal is remounted in the loop and flash cooled. In situ annealing techniques, where the cold stream is diverted and the crystal allowed to warm on the loop prior to flash cooling, are variations of annealing that appears to work best when large solvent channels are not present in the crystal lattice or the solvent content of the crystal is relatively low.

Aldose-Ketose Isomerases↗
PubMed1994

Bone crystal sizes: a comparison of transmission electron microscopic and X-ray diffraction line width broadening techniques.

Bone crystals are particularly small and hence estimating their sizes have proved to be difficult and values obtained inconsistent. Here we use rat bone crystals of different ages, as well as different synthetic carbonate apatite crystals, to compare two methods commonly used for determining bone crystal sizes. One method involves direct measurement of crystal lengths and widths, but not thicknesses, from transmission electron microscope (TEM) photographs of dispersed crystals. The second method utilizes X-ray diffraction line width broadening to estimate the average length of crystals. We conclude that line width broadening values tend to reflect crystal coherence lengths rather than the physical dimensions of the whole crystal. TEM measurements provide reliable estimates of average crystal lengths and widths and their ranges. Sample preparation procedures, however, cause breakage of the fragile crystals, which probably results in underestimates of in vivo crystal sizes.

Animals↗
PubMed2005

Compositional factors associated with calcium lactate crystallization in smoked Cheddar cheese.

Previous researchers have observed that surface crystals of calcium lactate sometimes develop on some Cheddar cheese samples but not on other samples produced from the same vat of milk. The causes of within-vat variation in crystallization behavior have not been identified. This study compared the compositions of naturally smoked Cheddar cheese samples that contained surface crystals with those of samples originating from the same vat that were crystal-free. Six pairs of retail samples (crystallized and noncrystallized) produced at the same cheese plant on different days were obtained from a commercial source. Cheese samples were 5 to 6 mo old at the time of collection. They were then stored for an additional 5 to 13 mo at 4 degrees C to ensure that the noncrystallized samples remained crystal-free. Then, the crystalline material was removed and collected from the surfaces of crystallized samples, weighed, and analyzed for total lactic acid, L(+) and D(-) lactic acid, Ca, P, NaCl, moisture, and crude protein. Crystallized and noncrystallized samples were then sectioned into 3 concentric subsamples (0 to 5 mm, 6 to 10 mm, and greater than 10 mm depth from the surface) and analyzed for moisture, NaCl, titratable acidity, L(+) and D(-) lactic acid, pH, and total and water-soluble calcium. The data were analyzed by ANOVA according to a repeated measures design with 2 within-subjects variables. The crystalline material contained 52.1% lactate, 8.1% Ca, 0.17% P, 28.5% water, and 8.9% crude protein on average. Both crystallized and noncrystallized cheese samples contained significant gradients of decreasing moisture from center to surface. Compared with noncrystallized samples, crystallized samples possessed significantly higher moisture, titratable acidity, L(+) lactate, and water soluble calcium, and significantly lower pH and NaCl content. The data suggest that formation of calcium lactate crystals may have been influenced by within-vat variation in salting efficacy in the following manner. Lower salt uptake by some of the cheese curd during salting may have created pockets of higher moisture and thus higher lactose within the final cheese. When cut into retail-sized chunks, the lower salt, higher moisture samples contained more lactic acid and thus lower cheese pH, which shifted calcium from the insoluble to the soluble state. Lactate and soluble calcium contents in these samples became further elevated at the cheese surface because of dehydration during smoking, possibly triggering the formation of calcium lactate crystals.

Calcium↗
PubMed1997

Comparative analysis of thaumatin crystals grown on earth and in microgravity.

The protein thaumatin was studied as a model macro-molecule for crystallization in microgravity-environment experiments conducted on two US Space Shuttle missions (USML-2 and LMS). In this investigation, we have evaluated and compared the quality of space- and earth-grown thaumatin crystals using X-ray diffraction analyses, and characterized them according to crystal size, diffraction resolution limit and mosaicity. Two different approaches for growing thaumatin crystals in the microgravity environment, dialysis and liquid-liquid diffusion, were employed as a joint experiment by our two investigative teams. Thaumatin crystals grown in a microgravity environment were generally larger in volume and the total number of crystals was less, relative to crystals grown on earth. They diffracted to significantly higher resolution and with improved diffraction properties, as judged by relative plots of I/sigma versus resolution. The mosaicity of space-grown crystals was significantly less than that of crystals grown on earth. Increased concentrations of protein in the crystallization chambers in microgravity led to larger crystals. The data presented here lend further support to the idea that protein crystals of improved quality can be obtained in a microgravity environment.

Crystallization↗