Apparent diffusion coefficients of benign and malignant salivary gland tumors. Comparison to histopathological findings.

OBJECTIVE: To evaluate the apparent diffusion coefficient (ADC) of benign and malignant salivary gland tumors in comparison to histopathological findings. MATERIALS AND METHODS: This study included 32 patients with a wide spectrum of major salivary gland tumors (17 benign, 15 malignant). Diffusion-weighted imaging (DWI) and ADC measurements were performed in all patients. The degrees of extracellular components (myxoid and chondroid matrices, microcysts and hyalinization), were histopathologically classified as mild, moderate and conspicuous. Comparisons were made of mean ADC values between benign and malignant tumors, and among tumors showing different degrees of extracellular components. RESULTS: Mean ADC values were 1.09+/-0.34 x 10(-3) mm(2)/s in malignant salivary gland tumors and 1.40+/-0.43 x 10(-3) mm(2)/s in benign salivary gland tumors. No significant difference in mean ADC values was found between benign and malignant tumors (P>0.05). However, mean ADC values increased with the degree of extracellular components. Mean ADC values were significantly different between mild and moderate degrees (P<0.05) of extracellular components, and between mild and conspicuous degrees (P<0.05), in both benign and malignant tumor groups. CONCLUSION: In this study, ADC values alone did not allow differentiation between benign and malignant salivary gland tumors. Comparison with histopathological findings suggests a correlation between the amount of extracellular components and mean ADC values in salivary gland tumors.

Structural analysis of leucine-rich-repeat variants in proteins associated with human diseases.

A number of human diseases have been shown to be associated with mutation in the genes encoding leucine-rich-repeat (LRR)-containing proteins. They include 16 different LRR proteins. Mutations of these proteins are associated with 19 human diseases. The mutations occur frequently within the LRR domains as well as their neighboring domains, including cysteine clusters. Here, based on the sequence analysis of the LRR domains and the known structure of LRR proteins, we describe some features of different sequence variants and discuss their adverse effects. The mutations in the cysteine clusters, which preclude the formation of sulfide bridges or lead to a wrong paring of cysteines in extracellular proteins or extracellular domains, occur with high frequency. In contrast, missense mutations at some specific positions in LRRs are very rare or are not observed at all.

[Transarterial chemoembolization (TAE) with degradable starch microspheres (DSM) in advanced hepatocellular carcinoma].

This study was undertaken to evaluate the clinical utility of chemoembolization using degradable starch microspheres (DSM), which resolve in a short period in patients with advanced hepatocellular carcinoma (HCC). Twenty-one patients underwent DSM chemoembolization 24 times. After a mixture of iodized oil and epirubicin was injected into the hepatic arteries, the patients were embolized with DSM alone 16 times. In the other 8 times, embolization was done in one hepatic lobe with DSM and in the other hepatic lobe with gelatin sponge (GS). There was no major complication related to chemoembolization. Tumor response (complete, partial, and minor responses) was found in 46% of patients after TAE. Tumor recurrence was found in 64% of responders after a mean period of 2.0 months. The response rate was significantly higher when chemoembolization was performed using both DSM and GS than when it was done with DSM alone (63% vs 37%, p < 0.04). Although the response rate after DSM-TAE is low, its anticancer effect is reinforced when used as an adjuvant therapy of GS-TAE.

Extracellular Toll-like receptor 2 region containing Ser40-Ile64 but not Cys30-Ser39 is critical for the recognition of Staphylococcus aureus peptidoglycan.

Toll-like receptor 2 (TLR2) and CD14 function as pattern recognition receptors for bacterial peptidoglycan (PGN). TLRs and CD14 possess repeats of the leucine-rich motif. To address the role of the extracellular domain of TLR2 in PGN signaling, we constructed CD14/TLR2 chimeras, in which residues 1-356 or 1-323 of CD14 were substituted for the extracellular domain of TLR2, and five deletion mutants of TLR2, in which the progressively longer regions of extracellular TLR2 regions were deleted. PGN induced NF-kappaB activation in HEK293 cells expressing TLR2 but not in cells expressing CD14/TLR2 chimeras. The cells transfected with a deletion mutant TLR2(DeltaCys30-Ile64) as well as TLR2(DeltaCys30-Asp160) and TLR2(DeltaCys30-Asp305) failed to respond to PGN, indicating the importance of the TLR2 region Cys(30)-Ile(64). Although TLR2(DeltaCys30-Ser39) conferred cell responsiveness to PGN, the cells expressing TLR2(DeltaSer40-Ile64) failed to induce NF-kappaB activation. In addition, NF-kappaB activity elicited by PGN was significantly attenuated in the presence of synthetic peptide corresponding to the TLR2 region Ser(40)-Ile(64). From these results, we conclude that; 1) CD14 cannot functionally replace the extracellular domain of TLR2 in PGN signaling; 2) the TLR2 region Cys(30)-Ser(39) is not required for PGN recognition; 3) the TLR2 region containing Ser(40)-Ile(64) is critical for PGN recognition.

Structure of the YSPTSPS repeat containing two SPXX motifs in the CTD of RNA polymerase II: NMR studies of cyclic model peptides reveal that the SPTS turn is more stable than SPSY in water.

The carboxyl-terminal domain of RNA polymerase II, which is rich in phosphorylation sites, contains 17--52 tandem repeats with the consensus sequence of the heptapeptide, YSPTSPS. The repeat unit of the heptapeptide has two SPXX motifs showing potential beta-turns, SPTS and SPSY. NMR studies were performed in water at pH 4.0 for two cyclic peptides containing one and two repeat units, cyclo-[C(1)R(2)D(3)Y(4)S(5)P(6)T(7)S(8)P(9)S(10)Y(11)S(12)R(13)D(14)C(15)] (peptide 1) and cyclo-[C(1)R(2)D(3)Y(4)S(5)P(6)T(7)S(8)P(9)S(10)Y(11)S(12)P(13)T(14)S(15)P(16)N(17)Y(18)S(19)R(20)D(21)C(22)] (peptide 2), which are cyclized with a disulfide bridge of two Cys residues at the N- and C-termini. SP in 1 and 2 are predominantly in trans form. The following NMR parameters were detected: (1) lower temperature coefficients of amide proton chemical shifts of T7 and S8 in 1, and Tx (T7 or T14), Sx (S8 or S15), Tz (T14 or T7) and Sz (S15 or S8) in 2, (2) significantly large deviation of H(alpha) chemical shifts from its random coil value (Delta H(alpha)) of Pro preceding the Thr (P6 in 1, and Px and Pz in 2), (3) relatively large (3)J(HNH alpha) coupling constants (>8.7 Hz) of T7 in 1 and Tx and Tz in 2, and (4) NOE (d(NN) (i, i+1)) connectivities between the amide protons of T7-S8 and S10-Y11 in 1, and Tx-Sx, S10-Y11, Tz-Sz, and N17-Y18 in 2, although two Pro-Thr-Ser segments in 2 (each of these are annotated by 'x' and 'z') in the first and second repeat units were not distinguishable. Comparison of the NMR parameters between the cyclic peptides and the corresponding linear peptides indicates that cyclization promotes structural stabilization in water. The present NMR data were consistent with the presence of a beta-turn at both SPTS and SPSY: S(5)P(6)T(7)S(8) and S(8)P(9)S(10)Y(11) in 1, and SPxTxSx, SPzTzSz, SP(9)S(10)Y(11), SP(16)N(17)Y(18) in 2. However, the structure of the SPTS segment is more stable than that of the SPSY segment. Conformations consistent with NMR parameters including NOE distances were obtained through molecular dynamics and energy minimization methods. These calculations yielded two stable conformers for the SPTS segment. One of the two corresponds to a type I beta-turn.

Contralateral pterional approach to a giant internal carotid-ophthalmic artery aneruysm: technical case report.

OBJECTIVE AND IMPORTANCE: The contralateral approach to internal carotid-ophthalmic artery aneurysms has been used in selected cases but has rarely been described for a giant internal carotid artery aneurysm. We report a case of giant aneurysm that was successfully clipped via the contralateral pterional approach. CLINICAL PRESENTATION: A 69-year-old woman was found to have two aneurysms: a small aneurysm at the left internal carotid-posterior communicating artery and a giant aneurysm at the right internal carotid-ophthalmic artery. INTERVENTION: A direct clipping operation was performed via the left pterional approach. After the small left internal carotid artery aneurysm was clipped, the contralateral giant aneurysm was further exposed and successfully clipped by use of the same approach via the prechiasmatic space. CONCLUSION: The contralateral pterional approach can be applied even for a giant aneurysm of the carotid-ophthalmic artery aneurysm when the neck of the aneurysm is small and when there is a space between the anterior wall of the aneurysm and the tuberculum sellae. Furthermore, such a giant aneurysm can be clipped more easily and safely via the contralateral approach without compromising visual functions. To our knowledge, this is the first reported case of a giant internal carotid-ophthalmic artery aneurysm approached contralaterally. The feasibility of this approach can be assessed preoperatively by three-dimensional computed tomographic angiography as well as by conventional cerebral angiography.

The binding of myristoylated N-terminal nonapeptide from neuro-specific protein CAP-23/NAP-22 to calmodulin does not induce the globular structure observed for the calmodulin-nonmyristylated peptide complex.

CAP-23/NAP-22, a neuron-specific protein kinase C substrate, is Nalpha-myristoylated and interacts with calmodulin (CaM) in the presence of Ca2+ ions. Takasaki et al. (1999, J Biol Chem 274:11848-11853) have recently found that the myristoylated N-terminal nonapeptide of CAP-23/NAP-22 (mC/N9) binds to Ca2+ -bound CaM (Ca2+/CaM). In the present study, small-angle X-ray scattering was used to investigate structural changes of Ca2+/CaM induced by its binding to mC/N9 in solution. The binding of one mC/N9 molecule induced an insignificant structural change in Ca2+/CaM. The 1:1 complex appeared to retain the extended conformation much like that of Ca2+/CaM in isolation. However, it could be seen that the binding of two mC/N9 molecules induced a drastic structural change in Ca2+/CaM, followed by a slight structural change by the binding of more than two but less than four mC/N9 molecules. Under the saturated condition (the molar ratio of 1:4), the radius of gyration (Rg) for the Ca2+/CaM-mC/N9 complex was 19.8 +/- 0.3 A. This value was significantly smaller than that of Ca2+/CaM (21.9 +/- 0.3 A), which adopted a dumbbell structure and was conversely 2-3 A larger than those of the complexes of Ca2+/CaM with the nonmyristoylated target peptides of myosin light chain kinase or CaM kinase II, which adopted a compact globular structure. The pair distance distribution function had no shoulder peak at around 40 A, which was mainly due to the dumbbell structure. These results suggest that Ca2+/CaM interacts with Nalpha-myristoylated CAP-23/NAP-22 differently than it does with other nonmyristoylated target proteins. The N-terminal amino acid sequence alignment of CAP-23/NAP-22 and other myristoylated proteins suggests that the protein myristoylation plays important roles not only in the binding of CAP-23/NAP-22 to Ca2+/CaM, but also in the protein-protein interactions related to other myristoylated proteins.

NMR studies of model peptides of PHGGGWGQ repeats within the N-terminus of prion proteins: a loop conformation with histidine and tryptophan in close proximity.

The N-terminal region of the prion protein from human and mouse contains five tandem repeats with the consensus sequence of PHGGGWGQ. NMR studies were performed in water for two cyclic peptides, cyclo-[C(1)R(2)Q(3)P(4)H(5)G(6)G(7)S(8)W(9)G(10)Q(11)R(12)D(13)C(14)] (C1) and cyclo-[C(1)R(2)D(3)P(4)H(5)G(6)G(7)G(8)W(9)G(10)Q(11)P(12)H(13)G(14)G (15)G(16)W(17)G(18)Q(19)R(20)D(21)C(22)] (C2), which are cyclized by a disulfide bridge between the Cys residues at the N- and C-termini, and for their corresponding linear peptides (L1 and L2) which are formed by reduction. The patterns of the C(alpha)H chemical shift difference of these four peptide mimetics were very similar to those observed for the tandem repeats of human prion protein reported by other researchers. The medium-range NOE connectivities were found between the C(beta)H of the H5 and the proton of the W9 side chain for L1. The corresponding NOEs were also observed in H5-W9 and H13-W17 of L2 with ambiguity. These observations indicate that histidine (i) is in close proximity to tryptophan (i+4). d(alphaN) (i,i+2) NOE connectivities were observed between W9 and Q11 of L1 and L2, and d(NN) (i,i+1) NOE connectivities were also observed for G10-Q11 of L1 and L2 and for G18-Q19 of L2. Significantly lower temperature coefficients of amide proton chemical shifts were obtained for Q11 and Q19 of L2 and C2. Structure calculations for L1 showed that HGG(G/S)W and (G/S)WGQ adopt a loop conformation and a beta-turn, respectively. These results strongly suggest that the tandem repeats within prion protein adopt a non-random structure.

Ca2+-bound calmodulin forms a compact globular structure on binding four trifluoperazine molecules in solution.

Small-angle X-ray scattering (SAXS), which determines the radius of gyration, R(g), and the pair distance distribution function, was used to investigate the conformational changes of calmodulin (CaM) on binding to an antagonist, trifluoperazine (TFP), with or without Ca(2+) in solution. We previously applied this SAXS method to CaM complexed with N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7) [Osawa, Kuwamoto, Izumi, Yap, Ikura, Shibanuma, Yokokura, Hidaka and Matsushima (1999) FEBS Lett. 442, 173-177] and found that the binding of two W-7 TFP molecules to one Ca(2+)-saturated CaM molecule induces structural changes from a 'dumb-bell' shape to a compact globular shape. We report here that the most compact globular shape whose size is consistent with that of the 1:2 Ca(2+)-saturated CaM-W-7 complex is formed by the binding of four TFP molecules to one Ca(2+)-saturated CaM molecule. Even in the absence of Ca(2+), the conformational changes of CaM occur on TFP binding, giving a slightly smaller R(g) than Ca(2+)-free CaM alone.

Super-motifs and evolution of tandem leucine-rich repeats within the small proteoglycans--biglycan, decorin, lumican, fibromodulin, PRELP, keratocan, osteoadherin, epiphycan, and osteoglycin.

Leucine-rich repeats (LRRs) with 20-30 amino acids in unit length are present in many proteins from prokaryotes to eukaryotes. The LRR-containing proteins include a family of nine small proteoglycans, forming three distinct subfamilies: class I contains biglycan/PG-I and decorin/PG-II; class II: lumican, fibromodulin, PRELP, keratocan, and osteoadherin; and class III: epiphycan/PG-Lb and osteoglycin or osteoinductive factor. Comparative sequence analysis of the 34 available protein sequences reveals that these proteoglycans have two types of LRRs, which we call S and T. The type S LRR is 21 residues long and has the consensus sequence of xxaPzxLPxxLxxLxLxxNxI. The type T LRR has 26 residues; its consensus sequence is zzxxaxxxxFxxaxxLxxLxLxxNxL. In both "x" indicates variable residue; "z" is frequently a gap; "a" is Val, Leu, or Ile; and I is Ile or Leu. These type S and TLRRs are ordered into two super-motifs--STT with about 73 residues in classes I and II and ST with about 47 residues in class III. The 12 LRRs in the small proteoglycans of I and II are best represented as (STT)4; the seven LRRs of class III as (ST)T(ST)2. Our analyses indicate that classes I/II and III evolved along different paths after the establishment of the precursor ST, and classes I and II also diverged after the establishment of the precursor (STT)4.

Molecular modeling of single polypeptide chain of calcium-binding protein p26olf from dimeric S100B(betabeta).

P26olf from olfactory tissue of frog, which may be involved in olfactory transduction or adaptation, is a Ca2+-binding protein with 217 amino acids. The p26olf molecule contains two homologous parts consisting of the N-terminal half with amino acids 1-109 and the C-terminal half with amino acids 110-217. Each half resembles S100 protein with about 100 amino acids and contains two helix-loop-helix Ca2+-binding structural motifs known as EF-hands: a normal EF-hand at the C-terminus and a pseudo EF-hand at the N-terminus. Multiple alignment of the two S100-like domains of p26olf with 18 S100 proteins indicated that the C-terminal putative EF-hand of each domain contains a four-residue insertion when compared with the typical EF-hand motifs in the S100 protein, while the N-terminal EF-hand is homologous to its pseudo EF-hand. We constructed a three-dimensional model of the p26olf molecule based on results of the multiple alignment and NMR structures of dimeric S100B(betabeta) in the Ca2+-free state. The predicted structure of the p26olf single polypeptide chain satisfactorily adopts a folding pattern remarkably similar to dimeric S100B(betabeta). Each domain of p26olf consists of a unicornate-type four-helix bundle and they interact with each other in an antiparallel manner forming an X-type four-helix bundle between the two domains. The two S100-like domains of p26olf are linked by a loop with no steric hindrance, suggesting that this loop might play an important role in the function of p26olf. The circular dichroism spectral data support the predicted structure of p26olf and indicate that Ca2+-dependent conformational changes occur. Since the C-terminal putative EF-hand of each domain fully keeps the helix-loop-helix motif having a longer Ca2+-binding loop, regardless of the four-residue insertion, we propose that it is a new, novel EF-hand, although it is unclear whether this EF-hand binds Ca2+. P26olf is a new member of the S100 protein family.

Time-resolved X-ray diffraction from tendon collagen during creep using synchrotron radiation.

In order to understand the molecular mechanism of relaxation phenomena in collagenous tissue, time-resolved, small-angle X-ray diffraction measurements were performed on bovine Achilles tendon collagen under creep. A tension-induced increase in the 67 nm period (D-period) was observed, and the strain in the D-period, epsilon D, was found to be almost proportional to the external force per unit cross-sectional area (average stress) of the specimen. With an increase in epsilon D, a change in the ratio of intensities of the third-order reflection peak of the D-period to that of the second-order peak was also observed. The increase in epsilon D was decomposed into three elementary processes of D-period deformation, which are presented on the basis of the Hodge-Petruska model: (1) molecular elongation, (2) increase in gap region, and (3) relative slippage of lateral adjoining molecules. Up to 8 MPa of average stress, the contribution to epsilon D originated mostly from only mode (1). At more than 10 MPa of average stress, modes (2) and (3) also contributed to fibril elongation. For epsilon D by molecular elongation (mode (1)), the time dependence of the D-period change in the immediate response region is a sharply shaped step function, while the contribution to epsilon D by molecular rearranging modes gives a slight creep nature at the immediate response region in the time dependence of epsilon D. Because this creep nature is observed at the immediate response, it is related qualitatively to the KWW function in a stress-relaxation modulus of collagenous tissue observed in an immediate response region (Sasaki et al. (1993). Journal of Biomechanics 26, 1369-1376). The elementary process of KWW-type relaxation is concluded to be related to the tension-induced molecular rearrangement within a D-period.

Evidence for calmodulin inter-domain compaction in solution induced by W-7 binding.

Small-angle X-ray scattering and nuclear magnetic resonance were used to investigate the structural change of calcium-bound calmodulin (Ca2+/CaM) in solution upon binding to its antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). The radius of gyration was 17.4+/-0.3 A for Ca2+/CaM-W-7 with a molar ratio of 1:5 and 20.3+/-0.7 A for Ca2+/CaM. Comparison of the radius of gyration and the pair distance distribution function of the Ca2+/CaM-W-7 complex with those of other complexes indicates that binding of two W-7 molecules induces a globular shape for Ca2+/CaM, probably caused by an inter-domain compaction. The results suggest a tendency for Ca2+/CaM to form a globular structure in solution, which is inducible by a small compound like W-7.

An automated method for the simultaneous determination of pravastatin and its main metabolite in human plasma by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry.

A new method for the determination of pravastatin, a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and its main metabolite (R-416) in human plasma using high-performance liquid chromatography/atmospheric pressure (negative ion) chemical ionization mass spectrometry (LC/APCI-MS) is described. Pravastatin and R-416 in human plasma were isolated using solid phase extraction technique and analyzed by LC/APCI-MS. Selected ion monitoring was employed for selectivity and sensitivity, which enabled the quantification over a range of 0.625-80 mg/mL with acceptable precision and accuracy. No derivatization was required for these polar molecules. The retention times of the pravastatin, R-416 and the internal standard (R-1437) were 2.1, 2.5 and 3.9 min, respectively, with a total analysis time of 5 min. This method was validated and compared with the automated gas chromatography/negative ion chemical ionization mass spectrometry procedure.

Small-angle X-ray scattering and computer-aided molecular modeling studies of 20 kDa fragment of porcine amelogenin: does amelogenin adopt an elongated bundle structure?

Amelogenins, which are major matrix constituents in the developing tooth, play a regulatory role in the process of enamel crystal formation. Porcine amelogenin with 173 amino acid residues is rich in proline, glutamine, leucine, and histidine. We utilized the small-angle X-ray scattering (SAXS) technique to examine the solution structure of porcine amelogenin. Samples used were two porcine amelogenins with apparent molecular weights of 20 kDa (amino acids 1 to 148) and 13 kDa (amino acids 46 to 148) on SDS-PAGE. Prior to SAXS measurements, the protein samples were dissolved in 2% (v/v) acetic acid to give a concentration range up to 10 mg/ml. Comparison between Rg (the overall radius of gyration) and Rc (the cross-sectional radius of gyration) revealed that the 20 kDa amelogenin exists in this solution as asymmetric particles with a length of about 15 nm, presumably corresponding of dimers. Based on these experimental data and computer-aided molecular modeling studies, we propose that the 20 kDa amelogenin adopts an elongated bundle structure which mainly consists of extended structures similar to polyproline II and/or beta-strand, interspersed with beta-turn or loop.

A hairpin-loop conformation in tandem repeat sequence of the ice nucleation protein revealed by NMR spectroscopy.

The 1H-NMR spectrum of a synthetic 24-residue peptide (A1-G-V-D-S-S-L-I-A-G-Y-G-S-T-Q-T-S-G-S-D-S-A-L-T24; INP24), comprising three repeats of the 8-residue consensus sequence of Pseudomonas syringae ice nucleation protein, was fully assigned using 2-dimensional (2D) NMR spectroscopy at 4 degrees C and 30 degrees C. Close proximity of the aliphatic protons between Leu7, Ile8, Ala9, and the ring-protons of Tyr11 was indicated from the observation of the inter-molecular nuclear Overhauser enhancement (NOE) effect. Hydrogen-bonding was strongly suggested for the NH group of Leu7 from its extremely low-temperature coefficient estimated from the temperature dependence of the chemical shift. These results indicate the formation of a hairpin-loop conformation constructed by a hexapeptide segment of INP24, -Leu7-Ile8-Ala9-Gly10-Tyr11-Gly12.

Three-dimensional structure of maize alpha-zein proteins studied by small-angle X-ray scattering.

alpha-zeins of maize (Zea mays) that are storage proteins contain nine or ten tandem repeats comprising of about 20 amino acids. Small-angle X-ray scattering (SAXS) of alpha-zeins was measured in 70% (v/v) aqueous ethanol containing beta-mercaptoethanol or without reagent in a protein concentration range of 2.0 to 40.0 mg/ml. The overall radius of gyration of whole particles, Rg, and the corresponding radius of gyration of the cross-section, Rc, of reduced alpha-zeins are 4.00 +/- 0.03 nm and 1.39 +/- 0.05 nm, respectively, in the 70% (v/v) aqueous ethanol containing 2% (v/v) beta-mercaptoethanol. Analyses using the Rg and Rc values indicate that reduced alpha-zeins exist as asymmetric particles with the length of about 13 nm in the solution. A structural model is developed under assumption that each of tandem repeats units forms single alpha-helix and they are joined by glutamine-rich 'turns' or loops, as employed by Argos et al., [Argos, O., Pedersen, K., Marks, M.D. and Larkins, B.A. (1982) J. Biol. Chem. 257, 9984-9990] and Garratt et al. [Garratt, R., Oliva, G., Caracelli, I., Leite, A. and Arruda, P. (1993) Proteins Struc. Func. Genet. 15, 88-99], and that the longest dimension of 13 nm comes from linear stacking of the anti-parallel helices of tandem repeat in the direction perpendicular to the helical axis. The resultant model is presented by an elongated prism-like shape with an approximate axial ratio of 6:1.

EF-hand motifs of alpha, beta and gamma isoforms of diacylglycerol kinase bind calcium with different affinities and conformational changes.

The three diacylglycerol kinase isoenzymes (DGK alpha, DGK beta and DGK gamma) cloned so far contain in common a tandem repeat of EF-hand motifs. However, the Ca2+ dependences of the DGK activities are known to be variable between isoenzymes, and the Ca(2+)-binding activities of these motifs have not been tested except for those present in DGK alpha. We therefore attempted to define the intrinsic properties of EF-hands occurring in the DGK isoenzymes. For this purpose we bacterially expressed and purified the EF-hand motifs (termed DKE forms) of the three DGKs. Equilibrium dialysis with the purified DKE forms showed that all of the expressed proteins could bind approx. 2 mol of Ca2+ per mol. However, the apparent dissociation constant (Kd) for calcium binding to alpha-DKE (9.9 microM) was an order of magnitude greater than those estimated for beta-DKE (0.89 microM) and gamma-DKE (0.40 microM). Experiments with 2-p-toluidinyl-naphthalene 6-sulphonate, a probe for hydrophobic regions of proteins, showed that the binding of Ca2+ to beta-DKE resulted in the exposure of hydrophobic amino acids, whereas hydrophobic regions of alpha-DKE and gamma-DKE were masked by the addition of Ca2+. Taken together, these results indicate that DGK alpha, DGK beta and DGK gamma possess EF-hand structures with intrinsic properties different from each other with respect to affinities for Ca2+ and Ca(2+)-induced conformational changes.

[Usefulness of functional image of dynamic computed tomography (FID-CT) on acute cerebral ischemic disease: correlation between parameters of dynamic CT and angiographical findings].

We reported the correlation between the angiographical findings and parameters of the functional images of dynamic CT (FID-CT) in ten patients with cerebral ischemic disease. Six of these ten patients with an abnormal area on corrected mean transit time (CM) and time to peak (TP) had complete occlusion of intracerebral main arteries with poor collateral cerebral blood flow or no collateral flow on the angiography. Four patients with prolonged time only in the TP area, but not in the CM area, had stenosis or occlusion of the main trunk with satisfactory anterograde cerebral blood flow on the angiography. FID-CT is easy to apply for clinical use and said to be useful to predict arterial trunk occlusion or infarction volume. It might also be helpful to get further information about the intracerebral circulation disorders in the acute stage of cerebral ischemic disease.