MR Imaging Detection of CNS Lesions in Tuberous Sclerosis Complex: The Usefulness of T1WI with Chemical Shift Selective Images.

BACKGROUND AND PURPOSE: CNS lesions of tuberous sclerosis complex are diagnosed mainly by T2WI, FLAIR, and sometimes T1WI with magnetization transfer contrast. The usefulness of T1WI with chemical shift selective images was recently reported in focal cortical dysplasia type IIb, which has histopathologic and imaging features similar to those of tuberous sclerosis complex. We investigated the usefulness of the T1WI with chemical shift selective images in detecting CNS lesions of tuberous sclerosis complex. MATERIALS AND METHODS: We retrospectively reviewed 25 consecutive patients with tuberous sclerosis complex (mean age, 11.9 [SD, 8.9] years; 14 males) who underwent MR imaging including T1WI, T1WI with magnetization transfer contrast, T1WI with chemical shift selective, T2WI, and FLAIR images. Two neuroradiologists assessed the number of CNS lesions in each sequence and compared them in 2 steps: among T1WI, T1WI with magnetization transfer contrast and T1WI with chemical shift selective images, and among T2WI, FLAIR, and T1WI with chemical shift selective images. We calculated the contrast ratio of the cortical tubers and of adjacent normal-appearing gray matter and the contrast ratio of radial migration lines and adjacent normal-appearing white matter in each sequence and compared them. RESULTS: T1WI with chemical shift selective images was significantly superior to T1WI with magnetization transfer contrast for the detection of radial migration lines and contrast ratio of radial migration lines. There was no significant difference between T1WI with chemical shift selective images and T1WI with magnetization transfer contrast for the detection of cortical tubers and the contrast ratio of the cortical tubers. Both T2WI and FLAIR were statistically superior to T1WI with chemical shift selective images for the detection of cortical tubers. T1WI with chemical shift selective images was significantly superior to T2WI and FLAIR for the detection of radial migration lines. CONCLUSIONS: The usefulness of T1WI with chemical shift selective images in detecting radial migration lines was demonstrated. Our findings suggest that the combination of T1WI with chemical shift selective images, T2WI, and FLAIR would be useful to evaluate the CNS lesions of patients with tuberous sclerosis complex in daily clinical practice.

Stereological quantification of olfactory receptor neurons in mice.

The total number of olfactory receptor neurons (ORNs) in the mouse main olfactory epithelium (MOE) was estimated using stereological sampling. Noses and skulls of male and female 8-week-old C57BL/6J mice were de-calcified, embedded in paraffin, cut into 10-µm-thick sections serially at 100-µm intervals, and processed for immunohistochemistry for the olfactory marker protein (OMP), a specific marker for ORNs. The number of OMP (+) receptor neurons was measured using an optical fractionator with the Stereo-Investigator software. The mean values of the total number of OMP (+) receptor neurons in the unilateral MOE were 5,140,000±380,000 in males and 5,210,000±380,000 in females, with no significant differences between the sexes. We concluded that the total number of ORNs in the unilateral MOE is approximately 5×10(6) in mice.

Catecholaminergic polymorphic ventricular tachycardia: electrocardiographic characteristics and optimal therapeutic strategies to prevent sudden death.

OBJECTIVE: To investigate the clinical outcome, ECG characteristics, and optimal treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT), a malignant and rare ventricular tachycardia. PATIENTS AND METHODS: Questionnaire responses and ECGs of 29 patients with CPVT were evaluated. Mean (SD) age of onset was 10.3 (6.1) years. RESULTS: The initial CPVT manifestations were syncope (79%), cardiac arrest (7%), and a family history (14%). ECGs showed sinus bradycardia and a normal QTc. Mean heart rate during CPVT was 192 (30) beats/min. Most cases were non-sustained (72%), but 21% were sustained and 7% were associated with ventricular fibrillation. The morphology of CPVT was polymorphic (62%), polymorphic and bidirectional (21%), bidirectional (10%), or polymorphic with ventricular fibrillation (7%). There was 100% inducement of CPVT by exercise, 75% by catecholamine infusion, and none by programmed stimulation. No late potential was recorded. Onset was in the right ventricular outflow tract in more than half the cases. During a follow up of 6.8 (4.9) years, sudden death occurred in 24% of the patients, 7% of whom had anoxic brain damage. Autosomal dominant inheritance was seen in 8% of the patients' families. beta Blockers completely controlled CPVT in only 31% of cases. Calcium antagonists partially suppressed CPVT in autosomal dominant cases. CONCLUSIONS: CPVT may arise in certain distinct areas but the prognosis is poor. The onset of CPVT may be an indication for an implanted cardioverter-defibrillator.

Non-invasive measurement of coronary flow reserve in children with Kawasaki disease.

OBJECTIVE: To investigate whether transthoracic Doppler echocardiography (TTE) can reliably measure the coronary flow reserve in the left anterior descending coronary artery in children with Kawasaki disease. DESIGN: Coronary flow velocity in the distal left anterior descending coronary artery was measured by TTE and was compared with that obtained by intracoronary Doppler guide wire. The ratio of maximum hyperaemia (intravenous administration of adenosine triphosphate, 160 microg/kg/min) to baseline peak (mean) diastolic coronary flow velocity in the distal artery was used as an estimate of coronary flow reserve. SETTING: University hospital. PATIENTS: 10 patients with significant left anterior descending coronary stenosis (> 70% diameter stenosis) (group A) in the proximal or middle portion of the artery and 14 patients (group B) without significant stenosis, all with Kawasaki disease documented by previous coronary angiography. RESULTS: The reduced hyperaemic coronary flow velocity in group A compared with group B resulted in a markedly lower coronary flow reserve, derived from both peak diastolic velocity and mean diastolic velocity by either technique of investigation. Multivariate analysis identified the best predictor of left anterior descending coronary artery stenosis to be a coronary flow reserve of < or = 2.2, derived from mean diastolic flow velocity measured using TTE (sensitivity 90%, specificity 100%, accuracy 96%). A good correlation was found between diastolic velocity derived values for coronary flow reserve measured using both TTE and Doppler guide wire (r = 0.92, p = 0.0001). CONCLUSIONS: Coronary flow reserve in the distal left anterior descending coronary artery can be accurately measured using TTE without any intravascular instrumentation in children with Kawasaki disease.

A novel alkaline endoglucanase from an alkaliphilic Bacillus isolate: enzymatic properties, and nucleotide and deduced amino acid sequences.

A highly alkaline endo-1,4-beta-glucanase (Egl) was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp. strain KSM-N252. The optimal pH for activity was as high as 10, and the optimal temperature was 55 degrees C. The molecular mass and isoelectric point were around 50 kDa and pH 4.2, respectively. The enzyme hydrolyzed carboxymethyl cellulose in a random fashion. Unlike previously reported Egls, the enzyme was highly active on p-nitrophenyl cello-oligosaccharides and acid-swollen cellulose, and its activity was stimulated by cellobiose at high concentrations. The entire gene for the enzyme contained a 1,476-bp single open reading frame encoding 492 amino acids, including a 29-amino-acid signal peptide. The mature enzyme (463 amino acids: 51,174 Da) exhibited moderate homology to other family 5 alkaline Egls. In the C-terminal region, a carbohydrate-binding module that belongs to family XII was repeated. Furthermore, four and six repeats of Pro-Pro-Ser/Thr-Glu/Asp-Pro-(Glu) were found immediately before the first and second carbohydrate-binding modules, respectively.

Noninvasive assessment of the early progression of atherosclerosis in adolescents with Kawasaki disease and coronary artery lesions.

OBJECTIVES: The severity of carotid arteriosclerosis, defined as increased intima-media thickness (IMT) visible with B-mode ultrasound, is correlated with risk factors for coronary heart disease. Little is known, however, about the relation of IMT, the stiffness of the common carotid artery, and the progression of atherosclerosis in children with Kawasaki disease (KD) and coronary artery lesions (CAL). DESIGN: We investigated the associations among total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglyceride, TC/HDL-C, ApoE genotype, glycohemoglobin, IMT, and arterial stiffness by high resolution ultrasound in 20 adolescents with KD and CAL (age: 16.6 +/- 4.1 years, males: 60%) and 20 sex- and age-matched healthy controls (Cont). RESULTS: No significant differences were observed in the values of body mass index and blood pressure between the 2 groups. Also no significant differences were observed in the levels of TC, LDL-C, HDL-C, triglyceride, TC/HDL-C within normal values, or the allele frequency of Apo E4 between the 2 groups. However, the values of IMT, stiffness, and glycohemoglobin in patients with KD were significantly higher than those in Cont (0.53 +/- 0.07 vs 0.46 +/- 0.05 mm, P <.05; 4.11 +/- 0.86 vs 2.94 +/- 0.91, P <.001; 5.0 +/- 0.4 vs 4.6 +/- 0.2%, P <.05). A significant relationship was observed between sex and the mean IMT (r = 0.46) in Cont; however, no significant correlation was found between mean IMT and other variables in either group. Although the arterial stiffness was strongly related to body mass index (r = 0.58) and systolic blood pressure (r = 0.55) in Cont, no significant relationship was observed between the stiffness and any variable tested in patients with KD. CONCLUSIONS: The less distensible carotid wall in patients with KD compared with that in Cont without major alteration of the lipid profile is likely to be secondary to the changes in arterial walls after a diffuse vasculitis involving noncoronary arteries. These results indicate that the coronary arteries may be predisposed to accelerated atherosclerosis in patients with KD and CAL.

A new high-alkaline and high-molecular-weight pectate lyase from a Bacillus isolate: enzymatic properties and cloning of the gene for the enzyme.

A pectate lyase (Pel; pectate transeliminase: EC4.2.2.2.), designated Pel-15H, was found in an alkaline culture of Bacillus sp. strain KSM-P15 and purified to homogeneity by sequential column chromatographies. The molecular weight of the enzyme determined by SDS-polyacrylamide gel electrophoresis was approximately 70,000 and the pI was around pH 4.6. Pel-15H randomly trans-eliminated polygalacturonate in the presence of Ca2+ ions, and the maximum activity was observed at pH 11.5 and at 55 degrees C in glycine-NaOH buffer. The gene for Pel-15H was cloned and sequenced, and the structural gene contained a 2,031-bp open reading frame that encoded 677 amino acids including a possible 28-amino-acid signal sequence. The mature enzyme (649 amino acids, molecular weight 69,550) showed very low similarity to Pels from Bacillus with 12.7-18.2% identity. Interestingly, part of the amino acid sequence of Pel-15H had fairly high similarity only to an N-terminal half of PelL and a C-terminal half of PeIX from Erwinia chrysanthemi 3937, and a C-terminal half of PeIX from E. chrysanthemi EC16 (approximately 35% identity for all).

Nucleotide and amino-acid sequences of a new-type pectate lyase from an alkaliphilic strain of Bacillus.

A pectate lyase (pectate transeliminase; EC 4.2.2.2), designated Pel-15E, was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp. strain KSM-P15. The purified enzyme had a molecular mass of approximately 33 kDa, as determined by SDS/PAGE, and a pI of approximately pH 9.2. Pel-15E exhibited optimum activity at pH 10.5 and 50-55 degrees C in glycine/NaOH buffer. Pel-15E had an absolute requirement for Ca2+ ions for manifestation of the enzymatic activity and trans-eliminated poly(galacturonic) acid, most likely by endo-type cleavage. A gene for the enzyme, which was cloned using the shotgun method and sequenced, contained a 960-bp ORF encoding 320 amino acids. The mature enzyme (286 amino acids, 32 085 Da) from the deduced amino-acid sequence showed quite low homology to known Pels from various microorganisms with 16.1-20.4% identity. Furthermore, we were not able to find any conserved regions in the sequence of Pel-15E when aligned with the sequences of other enzymes from the established Pel superfamily. However, Pel-15E had some regions that were homologous to PelA from Azospirillum irakense with 39.8% identity. Based on their amino-acid sequence homology, Pel-15E and PelA appear to belong to a new class of Pel family, although the enzymatic properties of both enzymes were quite different.

[Assessment of cardiac sympathetic nerve activity in children with chronic heart failure using quantitative iodine-123 metaiodobenzylguanidine imaging].

OBJECTIVES: Cardiac sympathetic nerve activity in children with chronic heart failure was examined by quantitative iodine-123 metaiodobenzylguanidine (MIBG) myocardial imaging in 33 patients aged 7.5 +/- 6.1 years (range 0-18 years), including 8 with cardiomyopathy, 15 with congenital heart disease, 3 with anthracycrine cardiotoxicity, 3 with myocarditis, 3 with primary pulmonary hypertension and 1 with Pompe's disease. METHODS: Anterior planar images were obtained 15 min and 3 hr after the injection of iodine-123 MIBG. The cardiac iodine-123 MIBG uptake was assessed as the heart to upper mediastinum uptake activity ratio of the delayed image (H/M) and the cardiac percentage washout rate (%WR). RESULTS: The severity of chronic heart failure was class I (no medication) in 8 patients, class II (no symptom with medication) in 9, class III (symptom even with medication) in 10 and class IV (late cardiac death) in 6. H/M was 2.33 +/- 0.22 in chronic heart failure class I, 2.50 +/- 0.34 in class II, 1.95 +/- 0.61 in class III, and 1.39 +/- 0.29 in class IV (p < 0.05). %WR was 24.8 +/- 12.8% in chronic heart failure class I, 23.3 +/- 10.2% in class II, 49.2 +/- 24.5% in class III, and 66.3 +/- 26.5% in class IV (p < 0.05). The low H/M and high %WR were proportionate to the severity of chronic heart failure. CONCLUSIONS: Cardiac iodine-123 MIBG showed cardiac adrenergic neuronal dysfunction in children with severe chronic heart failure. Quantitative iodine-123 MIBG myocardial imaging is clinically useful as a predictor of therapeutic outcome and mortality in children with chronic heart failure.

Serum amyloid A, cytokines, and corticosterone responses in germfree and conventional mice after lipopolysaccharide injection.

To determine why germfree mice are less susceptible to lipopolysaccharide (LPS) than conventional mice, we studied serum levels of serum amyloid A (SAA), tumor necrosis factor (TNF), interleukin 1 (IL-1), IL-6, and corticosterone in mice after treatment with LPS. A single injection of LPS caused an elevation of SAA, an acute-phase protein in the mouse, in both conventional and germfree IQI mice, and the response was significantly less in germfree mice. LPS-induced elevations of serum TNF, IL-1, and IL-6 levels were also significantly less in germfree mice, while serum corticosterone levels were greater in germfree mice than in conventional mice. These results suggest that the lower susceptibility to LPS and a smaller response of SAA elevation by LPS in germfree mice may result from less elevation in serum of these cytokines in these mice, which are known to mediate the acute phase response of SAA. High levels of serum corticosterone in germfree mice may be partly responsible for the lower responsiveness of these inflammatory cytokines to LPS in these mice.

One-stage repair of interrupted aortic arch and aortopulmonary window.

Interrupted aortic arch type A with aortopulmonary window was diagnosed in a 12-day-old neonate. A successful one-stage repair was undertaken through a midline sternotomy without circulatory arrest. The aortopulmonary window was closed through the anterior wall of communication between ascending aorta and main pulmonary artery with a patch. Position of the arterial cannula was changed during the repair, which made it possible to mobilize and expose the aortic arch for the completion of direct anastomosis.

Supernormal conduction in a case of Mobitz type II atrioventricular block.

A 52-year-old woman exhibited Mobitz type II atrioventricular block with right bundle branch block and 1:1 atrioventricular conduction at or slower than 80 beats/min. Electrophysiologic study revealed transient HV interval block followed by recovery from the block at shorter coupling intervals without prolongation of the H1H2 and H2V2 intervals, suggesting true supernormal conduction. Isoproterenol enhanced the supernormal conduction, with shortening of blocked intervals and recovery of atrioventricular conduction, while atropine caused their less marked enhancement. Linking (ie, retrograde concealment of the impulse to the distal His bundle region through the blocked right bundle branch) is considered a possible mechanism of supernormal conduction in this case.

Measurement of coronary flow reserve in children by transthoracic Doppler echocardiography.

Noninvasive measurement of coronary flow reserve was performed by transthoracic color Doppler echocardiography in 28 children with Kawasaki disease.

[Optimum protocol of technetium-99m tetrofosmin myocardial perfusion imaging for the detection of coronary stenosis lesions in Kawasaki disease].

The clinical usefulness of a new myocardial perfusion imaging agent, technetium-99m tetrofosmin, was assessed in 58 patients for a total of 76 times (mean age 9.7 years, 1-15 years) including 20 with and 26 without significant coronary stenotic lesions in Kawasaki disease. Pharmacological (dobutamine or adenosine triphosphate disodium) or exercise stress technetium-99m tetrofosmin single photon emission computed tomography was performed under stress and at rest on the same day. The sensitivity for detection of stress-induced perfusion defects by this method was 90% (18/ 20) and the specificity was 85% (22/26). The dose was 9.3 +/- 2.5 MBq/kg under stress and 18.7 +/- 5.6 MBq/kg at rest. No significant correlation was recognized between the dose and the image quality. Adequate image quality was provided by projection time 20-40 sec per frame. Increased liver accumulation was seen in 24% (18/76), especially in younger and pharmacological stress cases. The acquisition starting time after tetrofosmin injection was 58.4 +/- 18.7 min in the negative increased liver accumulation group and 43.7 +/- 18.3 min in the positive group (p < 0.01). We recommend that the following protocol is used for pediatric imaging. 1) The standard tetrofosmin dose is rougly 10 MBq/kg (upper limit 370 MBq) at stress and the double dose for the rest imaging. 2) SPECT projection time of 20-40 sec per frame. 3) In practice, the patient should be fasting prior to stress injection, and the imaging should be done 1 hour after eating. The rest injection should be done immediately thereafter, and the rest image should be obtained 1 hour later.

Dobutamine stress echocardiography for detection of coronary artery stenosis in children with Kawasaki disease.

OBJECTIVES: This study was designed to assess the feasibility and diagnostic accuracy of dobutamine stress echocardiography for detection of coronary artery stenosis in children with Kawasaki disease. BACKGROUND: Dobutamine stress echocardiography is valuable as an alternative test for detection of coronary artery disease in adult patients; however, its usefulness for children has been demonstrated only in limited cases. METHODS: Dobutamine stress echocardiography (up to 30 microgram/kg body weight per min) was performed in 50 patients at the convalescent stage of Kawasaki disease, including 26 patients with coronary sequelae documented by previous coronary angiography (sequelae group, 3 to 15 years old) and 24 patients with normal coronary arteries documented by echocardiography (normal group, 7 to 16 years old), who underwent quantitative coronary angiography on a separate day. Left ventricular regional wall motion divided into 16 segments was assessed in relation to the extent of coronary artery disease. A positive test response was defined as a new or worsened wall motion abnormalities. RESULTS: Significant coronary artery disease (> or = 50% diameter stenosis of major vessels) was present in 21 patients in the sequelae group. There was no significant difference in the maximal dose of dobutamine between the sequelae and normal groups ([mean +/- SD] 22.4 +/- 5.1 vs. 24.2 +/- 2.5 microgram/kg per min). Heart rate and systolic blood pressure were significantly increased (p < 0.01) at maximal dose of dobutamine compared with values at rest in both groups; consequently, the rate-pressure product exceeded 20,000 in 20 (40%) of the 50 patients during dobutamine infusion. Ten patients had self-limiting side effects; however, there were no serious complications from stress-induced ischemia. New wall motion abnormalities corresponding to the extent of coronary artery disease were detected in 19 of 21 patients in the sequelae group, whereas no wall motion abnormalities were detected in the normal group. Thus, the sensitivity and specificity of dobutamine stress echocardiography for the detection of coronary artery disease were 90% and 100%, respectively. CONCLUSIONS: We conclude that dobutamine stress echocardiography is a safe and accurate diagnostic method for detection of coronary artery stenosis in Kawasaki disease. Moreover, this is a possible alternative method for patients unable to exercise adequately, even if they are small children.

Application of the upstream region of a Bacillus endoglucanase gene to high-level expression of foreign genes in Bacillus subtilis.

A 0.4-kb ScaI-HpaI fragment, 199 bp upstream of the structural gene for alkaline endoglucanase, from the alkalophilic Bacillus sp. KSM-64, was found to be essential for the extracellular production of the enzyme by recombinant Bacillus subtilis cells. We constructed a new vector, pHSP64 (5.5 kb), using pHY300PLK and part of the 5' region of the endoglucanase that contained a possible promoter region. Using recombinant B. subtilis cells that carried this vector, very high production of two endoglucanases and of chloramphenicol acetyltransferase was done.

Construction, purification, and properties of a truncated alkaline endoglucanase from Bacillus sp. KSM-635.

Part of a 2.4-kb DNA fragment that encoded the amino-terminal 584 residues (65 kDa) of an alkaline endoglucanase from Bacillus sp. KSM-635 (941 amino acid residues; 105 kDa) was spontaneously deleted during subcloning of the fragment. The remaining 1.1-kb insert of the deleted plasmid encoded amino acids from Ala228 to Leu584 of the enzyme. However, Escherichia coli HB101 cells harboring this plasmid produced an active endoglucanase. After addition of a termination codon, TAA, immediately downstream of the codon for Leu584, the 1.1-kb fragment was inserted into an expression vector, pHSP64. The resultant plasmid was introduced into Bacillus subtilis ISW1214 for extracellular production of the truncated endoglucanase. The enzyme was then purified to homogeneity from a culture of the recombinant B. subtilis cells. Amino-terminal sequencing of the enzyme showed that the enzyme consisted of 7 amino acid residues encoded by the vector and 357 amino acid residues encoded by the truncated gene, with a molecular mass of 40.2 kDa. The purified enzyme was very active against carboxymethylcellulose and its pH and temperature profiles were almost identical to those of the enzyme produced by Bacillus sp. KSM-635.

A longitudinal study on electrophysiological properties of atrioventricular accessory pathways in infants and children.

In this study the electrophysiological developmental changes of accessory pathway (AP) properties in infants and children are examined. Intracardiac and/or transesophageal electrophysiological studies were performed on seven infants (group 1, mean age 2 months) and four children (group 2, mean age 9 y; study 1), and documented orthodromic atrioventricular tachycardia in all cases. At follow-up study (study 2), supraventricular tachycardia was induced in three of seven (43%) cases in group 1 and in all four cases in group 2. Changes in antegrade conduction properties of AP were documented by loss of pre-excitation (two of four cases in group 1) or change to intermittent pre-excitation (one of two cases in group 2).

A stable isotope-aided NMR study of the active site of an endoglucanase from a strain of Bacillus.

Heteronuclear single-quantum coherence two-dimensional NMR spectroscopy has been used to investigate the active site of endoglucanase K (46 kDa) from Bacillus sp. KSM-330, in which Trp are important for expression of the activity. Endoglucanase K, which was specifically labeled with [indole-2-13C]Trp, was prepared from recombinant Bacillus subtilis that carried the gene for this enzyme on an expression vector, pHSP-KC331. Twelve cross-peaks originating from the C-2 position of Trp residues of endoglucanase K were separately observed in 1H-13C heteronuclear single-quantum coherence spectrum, and six of the cross-peaks have been assigned site-specifically by using site-directed mutagenesis. The chemical shifts of the cross-peaks originating from Trp-174 and Trp-243 were affected by the addition of cellotriose that was used as a competitive inhibitor of the enzyme. On the basis of the NMR data obtained after chemical modification of the enzyme by N-bromosuccinimide, it appears that Trp-174 was oxidized first with retention of 56% of the original activity and Trp-243 was then oxidized with complete loss of activity. Substitution of Trp-174 or Trp-243 by Tyr residue caused a decrease in the specific activity of the enzyme to 49 or 8% of that of the wild-type enzyme, respectively. Km values of these mutant enzymes for p-nitrophenyl beta-D-cellotrioside increased to 5 and 8 times those of the wild-type enzyme, respectively, while kcat values of both of the mutant enzymes decreased to one-fifth of those of the wild-type enzymes. These results suggest that Trp-174 and Trp-243 play an important role in binding of the substrate and/or in the catalytic activity.

Site-directed mutagenesis of the putative active site of endoglucanase K from Bacillus sp. KSM-330.

The roles of one Glu and four Asp residues of endoglucanase K from Bacillus sp. KSM-330, which are conserved in all the endo-beta-glucanases in the family D, were analyzed by site-directed mutagenesis. The gene for endoglucanase K was mutated to replace Asp-154, Asp-191, Asp-193 or Asp-300 by Asn, or to replace Glu-130 by Gln in the encoded enzyme. Mutant and wild-type genes were separately expressed in Bacillus subtilis and the resultant enzymes were purified from the culture broth. All mutant enzymes exhibited the same mobility on SDS-polyacrylamide gel electrophoresis as the wild-type enzyme and gave similar circular dichroism spectra to that of the wild-type enzyme. Substitution of Glu-130, Asp-191, Asp-193 or Asp-300 significantly decreased the specific activity of the enzyme toward CM-cellulose. Kinetic analysis of the abilities of these mutant enzymes to liberate p-nitrophenol from p-nitrophenylcellotrioside revealed that all the mutant enzymes had very much lower kcat values than that of the wild-type enzyme, while the Km values of these mutant enzymes were almost the same as that of the wild-type enzyme. Of these Glu and Asp residues, Glu-130 and Asp-191 seem to be most likely to be catalytic residues because substitutions of these residues resulted in the lowest kcat values of the mutant enzymes.