Comparison of postoperative surface light scattering of different intraocular lenses.

AIMS: This retrospective study was conducted to compare postoperative surface scattering of four kinds of intraocular lens (IOL). METHODS: Sixty-seven eyes of 67 patients who had undergone cataract surgery were enrolled in this study. One of four IOLs was used in the patients; MA60BM in 17 patients (MA group), SA60AT in 17 patients (SA group), AR40 in 16 patents (AR group) and ClariFlex in 17 patients (CL group). Measurement of scattering from the anterior surface of the IOL was measured with area densitometry using a Scheimpflug camera (EAS-1000, Nidek, Aichi) for 3 years after the surgery. RESULTS: The density of IOL surface scattering increased starting 1 year after surgery and throughout the 3-year period in the MA group and starting at 6 months through 3 years in the SA group, whereas the density was stable in the AR and CL groups. The density of surface scattering in the MA and SA groups at 3 years after surgery was significantly higher than in the AR and CL groups. CONCLUSION: The surface scattering of MA60BM and SA60AT is higher than that of AR40 and ClariFlex. The grades of surface scattering differ among the manufacturers, even with the same acrylic material.

Higher-order wavefront aberration and letter-contrast sensitivity in keratoconus.

AIMS: To evaluate the relation between higher-order aberration of the eye and contrast sensitivity function in eyes with keratoconus. METHODS: In 22 eyes of 14 patients with keratoconus (age 30.5+/-8.4 years, means+/-SD) and 26 eyes of 13 normal controls (age 29.2+/-6.7 years), ocular higher-order wavefront aberration for a 6-mm pupil was measured with the Hartmann-Schack aberrometer (KR-9000 PW, Topcon). The root mean square (RMS) of third- and fourth-order Zernike coefficients was used to represent higher-order aberrations. The letter-contrast sensitivity was examined using the CSV-1000LV contrast chart (Vector Vision). RESULTS: In the keratoconus group, the letter-contrast sensitivity showed significant correlation with third-order (Spearman's correlation coefficient r=-0.736, P<0.001) and fourth-order aberrations (r=-0.464, P<0.05). There was borderline correlation between log MAR BSCVA and third-order (r=0.413, P=0.070) and fourth-order aberrations (r=0.394, P=0.086). In the normal group, the letter-contrast sensitivity had no significant correlation with third-order (r=-0.170, P=0.411) and fourth-order aberrations (r=-0.088, P=0.673), and log MAR best spectacle-corrected visual acuity (BSCVA) showed no correlation with third-order (r=0.063, P=0.762) and fourth-order aberrations (r=-0.282, P=0.165). CONCLUSIONS: In eyes with keratoconus, there is significant correlation between contrast sensitivity and ocular higher-order wavefront aberrations.

Influence of tilt and decentration of scleral-sutured intraocular lens on ocular higher-order wavefront aberration.

AIM: To investigate the influence of tilt and decentration of scleral-sutured intraocular lenses (IOLs) on ocular higher-order wavefront aberrations. METHODS: In 45 eyes of 36 patients who had undergone scleral suture fixation of posterior chamber IOL, tilt and decentration of IOLs were determined by Scheimpflug videophotography, and higher-order aberration for a 4-mm pupil was measured using the Hartmann-Shack aberrometer. In another 100 eyes of 100 patients after standard cataract surgery with posterior chamber IOL implantation, ocular higher-order aberration was measured. RESULTS: In eyes with scleral-sutured IOL, the mean (SD) tilt angle and decentration were 4.43 degrees (3.02 degrees ) and 0.279 (0.162) mm, respectively. Ocular coma-like aberration in the sutured IOL group was 0.324 (0.170) microm, which was significantly greater than that of the standard cataract surgery group (0.169 (0.061) microm, p<0.001, Student's t test). No significant difference was found in ocular spherical-like aberration between the sutured IOL group (0.142 (0.065) microm) and standard surgery group (0.126 (0.033) microm; p = 0.254). In the sutured IOL group, IOL tilt significantly correlated with ocular coma-like aberration (Pearson's correlation coefficient r = 0.628, p<0.001), but no significant correlation was found between IOL tilt and ocular spherical-like aberration (r = 0.222, p = 0.175). The IOL tilt did not correlate with corneal coma-like (r = 0.289, p = 0.171) and spherical-like (r = 0.150, p = 0.356) aberrations. The IOL decentration did not correlate with any higher-order aberrations. CONCLUSION: In eyes with scleral-sutured posterior chamber IOL, tilting of the lens induces considerable amount of ocular coma-like aberrations.

Modifying modal characteristics of sound fields by state feedback control.

State feedback control of a sound field is investigated for improving the irregular distribution of the natural frequencies at low frequencies. A state-space description of a sound field is derived from an inhomogeneous wave equation using the finite element method. The feedback control system is realized as a feedback filter between sensor microphones and control sources using a state estimator for a linear dynamic system. Pole allocation is employed for calculating the state feedback gain vector, such that the roots of the closed-loop system have the desired modal distribution. Computer simulations are performed to demonstrate the control achieved by distributing the lowest undamped natural frequencies in a uniform manner. As a result, the transfer functions with the state feedback control are modified so as to have peaks at identical intervals for the frequency range of interest. A control experiment in an enclosure is carried out with the same objective of control as modeled in the simulations. The experiment verifies that the desired modal distribution can be achieved by introducing the proposed state feedback control into a sound field.

The correlation between 99mTc-MIBI uptake and MIB-1 as a nuclear proliferation marker in glioma--a comparative study with 201Tl.

Technetium-99m methoxy-isobutylisonitrile (MIBI), like thallium-201 (201Tl), is a highly efficient agent for the diagnosis and monitoring of glioma tumors. Although 201Tl uptake is known to be partly associated with proliferative activity, little is known about the correlation between MIBI uptake and proliferation activity in gliomas. The current study was performed to assess the correlation between MIBI uptake and proliferative activities in gliomas, estimated by the monoclonal antibody to Ki-67 antigen (MIB-1) staining method. By comparing the results with those of 201Tl, we determined which tracer would be suitable for estimating proliferative activities. Twenty-four presurgical glioma patients (six with low-grade gliomas, five with anaplastic astrocytomas, and 13 with glioblastomas) were given MIBI and 201Tl SPECT. Early (10 min after injection) and delayed images (3 h after injection) were obtained for both MIBI and 201Tl scintigraphy. SPECT parameters, early ratio (ER), delayed ratio (DR), and retention index (RI) were obtained in both radiopharmaceuticals. All patients underwent subsequent surgical excision, and the specimens were immunostained for MIB-1. The proliferative activity was measured as a percentage positive nuclear area for MIB-1 (MI; MIB-1 index). To evaluate the relationship between the proliferative activity and SPECT parameters, we performed a correlation analysis. MI correlated with the MIBI uptake ratio (r = 0.75 for ER, and r = 0.7 for DR). Both DR and RI of 201Tl also correlated with MI, but weakly (r = 0.6 for DR, and. r = 0.59 for RI). There was no significant correlation between the MIB-1 index and the other parameters. MIBI-uptake parameters demonstrated a stronger positive correlation with the MIB-1 index than that of 201Tl. With the use of MIBI SPECT, we can estimate the proliferative activity of glioma noninvasively.

Effects of pterygium on corneal spherical power and astigmatism.

OBJECTIVE: To evaluate prospectively the corneal refractive status before and after pterygium surgery and its relationship with preoperative pterygium size. DESIGN: Prospective, nonrandomized, comparative (self-controlled) trial. PARTICIPANTS: One hundred thirty-six eyes undergoing primary pterygium removal surgery. MAIN OUTCOME MEASURES: Corneal spherical power, astigmatism, surface regularity index (SRI), and surface asymmetry index (SAI) before and after surgery, and the preoperative pterygium size. RESULTS: Before surgery, pterygium size significantly correlated with spherical power (Pearson's correlation coefficient, r = -0.370, P < 0.001), astigmatism (r = 0.600, P < 0.001), SRI (r = 0.367, P < 0.001), and SAI (r = 0.387, P < 0.001). The surgery significantly increased spherical power of the cornea, whereas astigmatism, SRI, and SAI were significantly decreased by the surgery (P < 0.01, paired t test with Bonferroni's correction of P value for multiple comparison). Surgically induced changes in spherical power (r = 0.598, P < 0.001) and astigmatism (r = 0.653, P < 0.001) significantly correlated with the preoperative pterygium size. Precise prediction of the magnitude of refractive changes based on the preoperative pterygium size was difficult. CONCLUSIONS: The presence of pterygium and its removal significantly influence the corneal refraction including spherical power, astigmatism, asymmetry, and irregularity, with the larger pterygium exerting the greater influence.

Studies on thermal aggregation of bovine serum albumin as a drug carrier.

The irreversible thermal aggregation rate and process of bovine serum albumin (BSA) were investigated by means of light scattering technique as a function of temperature. The increasing rate of particle radius was affected by the aggregation temperature, concentration and the presence of fatty acid. The particle radius was larger and the aggregation rate was faster for fatty acid free BSA at higher temperature and concentration. Two thermal aggregation processes were observed at relatively low temperature and concentration, both for fatty acid containing (C-BSA) and fatty acid free BSA (F-BSA). The first process proceeds by an inter-monomer aggregation mechanism, and the second process by inter-aggregates aggregation. The first process is represented by a power law as Rhapp proportional to t(z), which is diffusion limited cluster aggregation (DLCA).

A new assay method of gamma-glutamyltransferase with 4-aminobenzoate hydroxylase from Agaricus bisporus as a coupling enzyme.

We have developed a new ultraviolet spectrophotometric method for measurement of serum gamma-glutamyltransferase activity. The principle of the method is as follows. Using L-gamma-glutamyl-3-chloro-4-aminobenzoate as the donor substrate, 3-chloro-4-aminobenzoate formed upon transfer of the gamma-glutamyl moiety from the donor substrate to the acceptor substrate glycylglycine is stoichiometrically converted to 3-chloro-4-hydroxyaniline by 4-aminobenzoate hydroxylase from Agaricus bisporus, coupled with the oxidation of NADH to NAD+, and the resulting decrease in absorbance at 340 nm is monitored. The results obtained indicate that there is a good possibility to establish an ultraviolet spectrophotometric method for measurement of serum gamma-glutamyltransferase activity using L-gamma-glutamyl-3-chloro-4-aminobenzoate as the donor substrate and 4-aminobenzoate hydroxylase from Agaricus bisporus as a coupling enzyme.

Molecular cloning, enhancement of expression efficiency and site-directed mutagenesis of rat epidermal cystatin A.

A rat cystatin A cDNA clone was isolated from a lambda ZAP library representing newborn rat skin mRNA by screening with a synthetic oligonucleotide designed from amino acid sequence 15-23 of the cysteine proteinase inhibitor. The obtained clone contained a partial coding region of the inhibitor, lacking the 5'-untranslated region and coding sequence for the NH(2)-terminal 13 residues. The amino acid sequence deduced from the base sequence, Glu14-Phe103, coincided with that determined at the amino acid level. To obtain the recombinant cystatin A protein, the DNA was fused with a synthetic linker encoding its missing N-terminal 17 residues and introduced into an expression vector, pMK2. In Escherichia coli, however, the expression level of the semi-synthetic gene was low, 0. 5 mg of the purified recombinant protein per 1 liter culture being produced. Changing of the codon usage of the N-terminal region in a pET-15b expression system led to an increase in the yield depending on the instability of the putative secondary structure around an initiation codon of the mRNA. The expressed cystatin A showed identical characteristics with the authentic form except for the absence of the N-terminal acetyl blocking group. Using the expression system, two kinds of point mutation, the conservative Val54 in the first loop QxVxG region being changed to Lys and Glu, were introduced, but there was almost no effect on the inhibitory activity toward papain. This suggests that the conserved Val in the reactive site is not restricted and that the hydrophobicity of the position is not essential for the activity of rat cystatin A.

Lipid-binding and antimicrobial properties of synthetic peptides of bovine apolipoprotein A-II.

We previously showed that bovine apolipoprotein A-II (apoA-II) had antimicrobial activity against Escherichia coli and the yeast Saccharomyces cerevisiae in PBS. We have characterized here the active domain of apoA-II using synthetic peptides. A peptide corresponding to C-terminal residues Leu(49)-Thr(76) exhibited significant antimicrobial activity against E. coli in PBS, but not against S. cerevisiae. Experiments using amino-acid-substituted peptides indicated that the residues Phe(52)-Phe(53)-Lys(54)-Lys(55) are required for the activity. Peptide Leu(49)-Thr(76) induced the release of calcein trapped inside the vesicles whose lipid composition resembles that of E. coli membrane, suggesting that peptide Leu(49)-Thr(76) can destabilize the E. coli membrane. CD measurements showed that the alpha-helicity of peptide Leu(49)-Thr(76) increased from 3.5 to 36% by addition of the vesicles. When E. coli cells were incubated with peptide Leu(49)-Thr(76), some proteins were released to the external medium, probably owing to membrane destabilization caused by the peptide. In electron micrographs of E. coli cells treated with peptide Leu(49)-Thr(76), transparent nucleoids and granulated cytoplasm were observed. Amino acid substitutions, Phe(52)Phe(53)-->AlaAla (Phe(52, 53)-->Ala) in peptide Leu(49)-Thr(76) caused the loss of antimicrobial activity against E. coli, although protein-releasing activity was retained. Electron micrographs of the cells treated with peptide Leu(49)-Thr(76)(Phe(52,53)-->Ala) revealed morphological change only at the nucleoids. Therefore peptide Leu(49)-Thr(76) appears to primarily target the cytoplasm rather than the membrane of E. coli cells.

A new UV method for serum gamma-glutamyltransferase assay using recombinant 4-aminobenzoate hydroxylase as a coupling enzyme.

4-aminobenzoate hydroxylase (4ABH) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 4-aminobenzoate to 4-hydroxyaniline. For use as a clinical reagent, the gene encoding 4ABH from Agaricus bisporus was cloned by the RACE method. Also, the cDNA encoding 4ABH was expressed in Escherichia coli cells as a fusion protein with glutathione S-transferase (GST). The expressed GST-4ABH fusion protein (recombinant 4ABH) in the soluble fraction exhibits decarboxylative hydroxylation and additional NADH oxidation activities.We investigated a new ultraviolet spectrometric method for determining serum gamma-glutamyltransferase (gamma-GT) using recombinant 4ABH as a coupling enzyme. The principle of the method is as follows. Using gamma-glutamyl-3-choloro-4-aminobenzoate (L-gamma-glu-PAClBA) and glycylglycine as the donor and acceptor substrates, 3-choloro-4-aminobenzoate (PAClBA) is formed by the catalysis of serum gamma-GT. PAClBA is stoichiometrically converted to 3-choloro-4-hydroxyaniline (PHClA) and NAD(+) by 4ABH and NADH. However, NADH oxidation results in a high reagent blank, which is considered as a drawback for use as a clinical reagent. Using recombinant 4ABH, we examined the effects of pH and detergents on these two activities, and found that several detergents suppress the additional NADH oxidation activity with little or no effect on hydroxylation activity. The results indicate a promising approach to establishing an ultraviolet spectrophotometric method for determining serum gamma-GT activity using L-gamma-glu-PAClBA as the donor substrate and recombinant 4ABH as a coupling enzyme.

Involvement of parathyroid hormone-related peptide in cell proliferation activity of human uterine leiomyomas.

Uterine leiomyomas develop from uterine smooth muscle cells, which are known to be regulated by estrogen and other growth factors. The purpose of this study was to investigate the role of expression of parathyroid hormone related-peptide (PTHrP) in the growth of uterine leiomyomas treated or untreated with gonadotropin-releasing hormone agonist (GnRH-a). Thirty-nine leiomyoma tissues were obtained from 36 patients who had been treated with GnRH-a (n=10) or without GnRH-a (n=29). The intensity of PTHrP immunostaining was categorized into three grades; "negative", "weakly positive", and "positive". Leiomyoma cell growth was estimated by the proliferating cell nuclear antigen (PCNA) labeling index (LI) with an image analyser. We also investigated the correlation between PTHrP expression and cell proliferation or histopathological findings. In the GnRH-a-untreated group, LI of the PTHrP "positive" group was significantly higher than that of the PTHrP "negative" group, but the intensity of PTHrP immunostaining did not correlate with LI in the GnRH-a-treated group. PTHrP expression did not correlate with histological findings or clinical parameters (age and phase of menstrual cycle) in either the GnRH-a-treated or the -untreated group. In addition, the expression of mRNA for PTHrP and its receptor was detected in leiomyomas by reverse transcriptase-polymerase chain reaction (RT-PCR). Our results indicate that the expression of PTHrP in leiomyomas correlated positively with cell growth in the GnRH-a-untreated group, suggesting that PTHrP may act as a local cell growth modifier in an autocrine/paracrine fashion on uterine leiomyomas.

Deletion of Ala144-Lys145 in Thermus thermophilus inorganic pyrophosphatase suppresses thermal aggregation.

The regions contributing to the thermostability of inorganic pyrophosphatase (PPase, EC 3.6.1.1) from Thermus thermophilus (Tth) were deduced in our previous study by random chimeragenesis, one of them being estimated to be Ala144-Lys145 [Satoh, T., Takahashi, Y., Oshida, N., Shimizu, A., Shinoda, H., Watanabe, M., and Samejima, T. (1999) Biochemistry 38, 1531-1536]. Therefore, we investigated the contributions of these two residues in Tth by preparing a deletion mutant (del.144-145 mutant) of Tth PPase. We examined its thermostability in terms of the CD and fluorescence spectra, and the thermal change in the enzymatic activity. The thermostability of the enzymatic activity of the del.144-145 mutant was similar to that of the wild type Tth PPase, whereas this mutant was more stable against heating. Furthermore, we compared the thermal aggregation of the wild type with that of the del.144-145 mutant. We found that the thermal aggregation of the mutant was reduced relative to that of the wild type. Moreover, the molecular weight of the mutant after heating at 90 degrees C was higher than that of the unheated one, whereas the wild type aggregated under the same conditions. Therefore, we can conclude that although the Ala144-Lys145 residues in Tth PPase may partly cause thermal aggregation, the deletion of these residues may stabilize the Tth PPase molecule structurally against heating and suppress thermal aggregation.

Site-directed mutagenesis of a possible type 1 copper ligand of bilirubin oxidase; a Met467Gln mutant shows stellacyanin-like properties.

In our previous paper, we reported a mutant of recombinant Myrothecium verrucaria bilirubin oxidase, in which the Met467 residue was replaced by Gly [Shimizu, A. et al. (1999) Biochemistry 38, 3034-3042]. This mutant displayed a remarkable reduction in enzymatic activity and an evident decrease in the intensity of the absorption band around 600 nm (type 1 charge transfer transition). In this study, we report the preparation of three Met467 mutants (Met467Gln, Met467His, and Met467Arg) and characterize their enzymatic activities, midpoint potentials, and absorption and ESR spectra. Met467His and Met467Arg show no enzymatic activity and a great reduction in the intensity of the absorption band around 600 nm. Furthermore, their ESR spectra show no type 1 copper signal, but only a type 2 copper signal; however, oxidation by ferricyanide caused the type 1 copper signal to appear. On the other hand, Met467Gln as expressed shows both type 1 and type 2 copper signals in its ESR spectrum, the type 1 copper atom parameters being very different from usual blue copper proteins but very similar to those of stellacyanin. The enzymatic activity of the Met467Gln mutant for bilirubin is quite low (0.3%), but the activity for potassium ferrocyanide is similar (130%) to that of the wild type enzyme. These results indicate that Met467 is important for characterizing the features of the type 1 copper of bilirubin oxidase.

Myrothecium verrucaria bilirubin oxidase and its mutants for potential copper ligands.

Bilirubin oxidase (EC:1.3.3.5) purified from a culture medium of Myrothecium verrucaria MT-1 (authentic enzyme) catalyzes the oxidation of bilirubin to biliverdin in vitro and recombinant enzyme (wild type) was obtained by using an overexpression system of the bilirubin oxidase gene with Aspergillus oryzae harboring an expression vector. The absorption and ESR spectra showed that both bilirubin oxidases are multicopper oxidases containing type 1, type 2, and type 3 coppers similar to laccase, ascorbate oxidase, and ceruloplasmin. Site-directed mutagenesis has been performed for the possible ligands of each type of copper. In some mutants, Cys457 --> Val, Ala, His94 --> Val, and His134.136 --> Val, type 1 and type 2 copper centers were perturbed completely and the enzyme activity was completely lost. Differing from the holoenzyme, these mutants showed type 3 copper signals. However, the optical and magnetic properties characteristic of type 1 copper were retained even by mutating one of the type 1 copper ligands, i.e., a mutant, Met467 --> Gly, showed a weak but apparent enzyme activity. A double mutant His456.458 --> Val had only type 1 Cu, showing a blue band at 600 nm (epsilon = 1.6 x 10(3)) and an ESR signal with very narrow hyperfine splitting (A parallel = 7.2 x 10(-)3 cm-1). Since the type 2 and type 3 coppers are not present, the mutant did not show enzyme activity. These results strongly imply that the peculiar sequence in bilirubin oxidase, His456-Cys457-His458, forms an intramolecular electron-transfer pathway between the type 1 copper site and the trinuclear center composed of the type 2 and type 3 copper sites.

A chimeric inorganic pyrophosphatase derived from Escherichia coli and Thermus thermophilus has an increased thermostability.

Factors contributing to the thermostability of inorganic pyrophosphatase (PPase) were investigated by examining chimeric PPases from Escherichia coli and Thermus thermophilus (Tth). Two chimeric PPase genes, T1-135E (residues 1-135 from the N terminus are comprised of Tth PPase and residues 136-173 are derived from the C terminus of E. coli PPase) and T1-149E [residues 1-149 from the N terminus are from Tth PPase and the rest (150-175) are from E. coli PPase], were constructed by random chimeragenesis. After the genes were overexpressed in the E. coli BL21(DE3) strain and the expression products were purified, we compared the characteristics of these chimeric PPases with those of the parental PPases. We found that the two chimeras had higher activity than either parent PPase at the optimum temperature. We also examined thermal stability in terms of CD spectra, fluorescence spectra, and thermal changes in enzyme activity. The results revealed that the thermal stability of T1-149E is similar to that of Tth PPase, but T1-135E is much more stable. This suggests that the four residues that are different between T1-135E and T1-149E may be critical for thermostability between the two chimeras. By comparing the three-dimensional structures of Tth and E. coli PPases, we deduced that the following two factors may contribute to differences in thermostability. (1) Two residues (Thr138 and Ala141 in the Tth PPase and His140 and Asp143 in the E. coli PPase) in the vicinity of the trimer-trimer interface were different. (2) The Ala144-Lys145 loop in the Tth PPase was deleted in the E. coli PPase and also in the T1-135E chimera. Therefore, we conclude that T1-135E was thermostabilized by these two factors, and also, the Tth PPase moiety may contribute to the structural integrity of the chimeric enzymes.

Primary structure, expression, and site-directed mutagenesis of inorganic pyrophosphatase from Bacillus stearothermophilus.

The complete primary structure of inorganic pyrophosphatase [EC 3.6. 1.1] from Bacillus stearothermophilus (ATCC 12016) was determined at the amino acid level by automated Edman degradation. The subunit of the enzyme consists of 164 amino acid residues with a calculated molecular mass of 18,796. The amino acid sequence of the enzyme is almost identical to that of thermophilic bacterium PS-3. Based on the determined primary structure, a PCR-amplified semi-synthetic gene was constructed and expressed in Escherichia coli JM109. The recombinant Bst. PPase showed the same characteristics and activity as the authentic enzyme, and exhibits higher thermostability than the E. coli enzyme. Furthermore, we prepared tyrosine-substituted variants by site-directed mutagenesis to elucidate the role of two highly conserved tyrosines (Y46 and Y130). As a result, two variants, Y46F and Y130F, lost most of their enzyme activity, whereas their conformations were unaffected. However, the wild-type and two variants exhibited different thermostability behaviors in the presence or absence of Mg2+. Therefore, these tyrosines may contribute to the structural integrity of the active site of the enzyme.

Hydrophobic interactions of Val75 are critical for oligomeric thermostability of inorganic pyrophosphatase from Bacillus stearothermophilus.

To determine the role of Val75 in the oligomeric structure of trimeric inorganic pyrophosphatase (PPase) [EC 3.6.1.1] from Bacillus stearothermophilus (Bst.), we used site-directed mutagenesis to prepare variants in which Val75 was replaced by Ala, Phe, Leu, Ile, Lys, Gln, and Asp. As a result, the variants in which valine is replaced by hydrophobic residues such as Ala, Phe, Leu, and Ile (V75A, F, L, and I) show almost the same level of enzyme activity and thermostability as the wild type enzyme, whereas variants with hydrophilic residue replacements such as Lys, Gln, and Asp (V75K, Q, and D) showed gross reductions in enzyme activity and thermostability. The dissociation of V75K and V75D from trimer to monomers occurred rapidly as the temperature rose, while V75F, V75L, and V75I dissociated more slowly than the wild type. There was no particular effect of heat treatment on the dissociation of V75A or V75Q, but these variants were slightly dissociated even in the native state. Thus, we conclude that Val75 may locate at the interface between the monomers and its hydrophobic interactions with its surroundings may play a key role in the thermostability and oligomeric subunit interactions of the enzyme.

Molecular cloning, expression, and site-directed mutagenesis of inorganic pyrophosphatase from Thermus thermophilus HB8.

The genomic DNA encoding the inorganic pyrophosphatase from an extremely thermophilic bacterium, Thermus thermophilus HB8 (ATCC27634), was isolated by colony hybridization with a probe designed as a part of gene amplified by the PCR method, which was derived from the partial amino acid sequence of the enzyme. The DNA was cloned into a plasmid vector, pUC118, after digestion with BamHI. The inserted nucleotide fragment was about 1.8 kbp in length and the nucleotide sequence included a 525 bp open reading frame. The deduced amino acid sequence was completely identical with that of the enzyme determined by automated Edman analysis of peptide fragments isolated from digests obtained with Staphylococcus aureus V8 protease and Achromobacter protease I, and also from products obtained on chemical cleavage with cyanogen bromide and 70% formic acid. The subunit of this enzyme is composed of 174 amino acid residues with a calculated molecular weight of 19,084. Then, the gene was overexpressed in Escherichia coli BL21 (DE3) using a plasmid vector, pET15b, system. The recombinant enzyme was fully active, and exhibited higher thermostability than the E. coli enzyme. Amino acid residues located on the surface of the recombinant enzyme were determined by means of limited proteolysis, and the results revealed that the environment of Lys residues is almost the same as the crystal structure reported previously [Teplyakov, A. et al. (1994) Protein Sci. 3, 1098-1107]. Furthermore, the roles of two tryptophan residues were investigated by site-directed mutagenesis, which indicated that they may be responsible for the structural integrity and thermostability.

[Experience in ESWL monotherapy using Lithostar--comparative study on the efficacy of standard-tube with C-tube].

Between October 1990 and April 1996, we treated 892 upper urinary tract stones with ESWL therapy using Lithostar (Siemens Medizinische Technik, Erlangen, Germany). In March 1993, the coupling head of lithotriptor was upgraded from "standard-tube" to "C-tube". The C-tube has approximately two times or more destructive energy and intensity than the standard-tube. In this study, we analyzed the clinical results according to type of coupling head in 713 cases treated by ESWL monotherapy and evaluated 3 months after the initial treatment. The overall success rate at 3 months after ESWL was 85% in the standard-tube cases and 93% in the C-tube cases; the stone-free rate was 72% and 82%, respectively. There were no cases in which had to be discontinued due to ESWL severe side effects. However, in 66.1% of the C-tube cases and 31.1% of the standard-tube cases, a sufficient destructive intensity could not be used because of pain. Within a group that received sufficient destructive intensity, the C-tube was able to reduce both duration of the procedure and the number of shots to two thirds of that of the standard-tube.