Crystal structure of alkaline cellulase K: insight into the alkaline adaptation of an industrial enzyme.

The crystal structure of the catalytic domain of alkaline cellulase K was determined at 1.9 A resolution. Because of the most alkaliphilic nature and it's highest activity at pH 9.5, it is used commercially in laundry detergents. An analysis of the structural bases of the alkaliphilic character of the enzyme suggested a mechanism similar to that previously proposed for alkaline proteases, that is, an increase in the number of Arg, His, and Gln residues, and a decrease in Asp and Lys residues. Some ion pairs were formed by the gained Arg residues, which is similar to what has been found in the alkaline proteases. Lys-Asp ion pairs are disfavored and partly replaced with Arg-Asp ion pairs. The alkaline adaptation appeared to be a remodeling of ion pairs so that the charge balance is kept in the high pH range.

Identification of thermostabilizing residues in a Bacillus alkaline cellulase by construction of chimeras from mesophilic and thermostable enzymes and site-directed mutagenesis.

An alkaliphilic Bacillus sp. strain, KSM-64, produces a mesophilic alkaline endo-1,4-beta-glucanase that is suitable for use in detergents. The deduced amino acid sequence of the enzyme showed very high homology to that of a thermostable alkaline enzyme from alkaliphilic Bacillus sp. strain KSM-S237. Analysis of chimeric enzymes produced from the genes encoding the mesophilic and thermostable enzymes suggested that the lysine residues at positions 137, 179, and 194 are responsible for their thermal stabilization. Replacing the corresponding Glu137, Asn179, and/or Asp194 with lysine by site-directed mutagenesis made the mesophilic enzyme more thermostable. Analyses of the hydrophilicity of deduced amino acid sequences and isoelectric focusing of the modified enzymes suggested that these three specific lysine residues and their replacements are all located on the surface of the enzyme molecule. This fact further suggested that specific ionic interaction is involved in the thermal stabilization of the enzyme.

Detection of nucleoprotein gene of Sendai virus in the lungs of rats by touchdown nested reverse transcription polymerase chain reaction.

The nucleoprotein (NP) gene of Sendai virus was detected by touchdown nested reverse transcription polymerase chain reaction (RT-PCR) in the lungs of a rat presented with respiratory illness and high serum ELISA titer to Sendai virus. This method seemed to be of value in controlling infection in laboratory rodents.

[Significance of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) activity in breast cancer tissue].

TS, DPD, uridine phosphorylase and thymidine phosphorylase are enzymes involved in the metabolism of the anticancer drug pyrimidine fluoride. In this study, levels of these enzymes were measured in 47 women with primary breast cancer. These enzyme levels were then compared to levels determined from breast cancer patients who received either preoperative chemotherapy or nothing, in order to determine whether they might predict clinical outcome. The TS inhibition rate was significantly higher (p < 0.05) in patients receiving preoperative chemotherapy (20.4 +/- 13.3%) than in the untreated group (11.4 +/- 9.8%). No other significant differences in activity were noted between the treated and untreated groups for any of the other enzymes studied. The activity of each enzyme at the tumor site and the tumor/normal (T/N) ratio were also compared between patients with and without recurrence. The TS inhibition rate at the tumor site was lower in recurring cases than in non-recurring cases, and the T/N ratio tended to be higher for DPD in patients with recurrences. These findings indicate that the TS inhibition rate and DPD activity may be useful predictors for early recurrence of breast cancer following surgery.

[University education in geriatrics--present status and future prospects of gerontology and geriatrics education in pathology].

The increase in the number and proportion of the elderly in Japan over the last 30 years has been faster than that in any other country. One of the measures we are compelled to take to deal with this drastic change in medicosocial circumstances is reform of the medical school curriculum. However, the necessary reforms are being implemented slowly and are still insufficient. We surveyed the present status of gerontology and geriatrics education in pathology, and the understanding, interest, and opinions on this matter among professors of pathology. Questionnaires were sent to 148 professors of pathology in 80 medical schools. Responses were received from 84 professors (57%) at 64 medical schools (80%). Of the 11 medical schools with a department of geriatrics 10(90%) included gerontology in the curriculum. In contrast, 43(80%) of the 53 remaining schools did not include gerontology in the curriculum, although education in geriatrics and gerontology has been given as part of pathology lectures in almost all medical schools. Many professors want to establish a department of geriatrics in their school, but feel it will be difficult because of lack of money and higher priority given to other fields. As other hindrances, most of the respondents noted the lack of money and higher priority given to other fields. As other hindrances, most of the respondents noted the lack of a good textbook of gerontology, ambiguity in the concept of the field, and the immaturity of gerontology as a science. Another major problem noted was uncertainty regarding the status of geriatrics as a clinical specialty. One professor mentioned that promotion of aging research would be the best way to solve these problems.

[University education in geriatrics. Present status and future plans of universities regarding the development of a program in geriatrics].

Because the number of people who reach an advanced age has been increasing at an unprecedented rate in Japan, geriatricians are expected to play a central role in health care for the elderly. However, only 16 out of 80 medical schools (20 percent) now have departments of geriatrics for undergraduate education. To develop undergraduate education in the field of geriatrics, a survey was sponsored by the Research Projects on Aging and Health (Health Science Research Grant the Ministry of Health and Welfare of Japan). A questionnaire regarding the present status and future plans of the university about a program in geriatrics, was sent to deans of medical faculties or vice-presidents of medical schools. The questionnaire included questions about current status and future plans regarding undergraduate geriatric education, the presence of a department or clinic of geriatrics, educational requirements in the field of geriatrics, opportunities for practice, institutions of practice, research on geriatrics, and other suggestions. The response rate was 93.7 percent (74/79). Departments or clinics of geriatrics had been established in 15 institutions (20.3 percent) and were planned in 18 (24.3 percent). Undergraduate education in geriatrics was considered necessary in 73 schools (98.7 percent) and indispensable as an obligatory subject in 56 (75.7 percent). Clinical practice was considered more important and effective than lectures in 50 schools (63.3 percent). Coordinated lectures on basic biomedical gerontology (such as mechanism of aging) and geriatric medicine for chronic degenerative diseases such as senile dementia were considered essential to the curriculum. In practicing geriatrics, experience in providing medical care to aged patients as well as social support and a welfare system for the aged is emphasized. Institutions, nursing homes, and geriatric hospitals outside medical schools be easily accessible. It was generally agreed that geriatrics should be taught in advanced classes. In conclusion, medical schools in Japan regard undergraduate education in geriatrics as necessary and agree on the optimal curriculum, but it is not universally implemented.

Purification and properties of mangano-superoxide dismutase from a strain of alkaliphilic Bacillus.

A mangano-superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from a strain of alkaliphilic Bacillus for the first time. The purified protein, with an isoelectric point of pH 4.5, had a molecular mass of approximately 50 kDa and consisted of two identical subunits (25 kDa). The N-terminal amino acid sequence was Ala-Tyr-Lys-Leu-Pro-Glu-Leu-Pro-Tyr-Ala-Ala-Asn-Ala-Leu-Glu-Pro-His-Ile- Asp-Glu-Ala. The optimum pH and temperature for the reaction were 7.5 and 35 degrees C, respectively. The properties of the superoxide dismutase were compared with those of the enzyme from thermophilic Bacillus stearothermophilus.

Profile analysis of chondroitin sulfates in human urine and serum.

A highly sensitive high-performance liquid chromatographic method with fluorometric postcolumn labeling using 2-cyanoacetamide was developed for the profile analysis of chondroitin sulfates (ChS) in normal human urine and serum. Over-sulfated disaccharide units such as di- or trisulfated unsaturated disaccharides in urine were estimated and unsaturated 6-sulfated disaccharide (delta Di-6S) was found as a major component from ChS in urine, although only small amounts of delta Di-6S from ChS were present in serum.

Deduced amino acid sequence and possible catalytic residues of a thermostable, alkaline cellulase from an Alkaliphilic bacillus strain.

Alkaliphilic Bacillus sp. strain KSM-S237 (a relative of Bacillus pseudofirmus) produces a thermostable, alkaline endo-1,4-beta-glucanase (Egl). The entire gene for the enzyme harbored a 2,472-bp open reading frame (ORF) encoding 824 amino acids, including a 30-aminoacid signal peptide. The deduced amino acid sequence of the mature enzyme (794 amino acids, 88,284 Da) showed very high similarity to those of family 5 mesophilic, alkaline Egls from some alkaliphilic bacilli. The enzyme had a region similar to a novel cellulose binding domain proposed for an Egl (EngF) from Clostridium cellulovorans. Expression of the Bacillus Egl gene in Bacillus subtilis resulted in high carboxymethy cellulase activity (2.0 g/l) in the culture broth, concomitant with the appearance of a protein band on an SDS gel at 86 kDa. Site-directed mutagenesis delineated the importance of Arg111, His151, Glu190, His262, Tyr264, and Glu305 in catalysis and/or substrate binding of the enzyme.

Highly alkaline pectate lyase Pel-4A from alkaliphilic Bacillus sp. strain P-4-N: its catalytic properties and deduced amino acid sequence.

The gene for a highly alkaline pectate lyase, Pel-4A, from alkaliphilic Bacillus sp. strain P-4-N was cloned, sequenced, and overexpressed in Bacillus subtilis cells. The deduced amino acid sequence of the mature enzyme (318 amino acids, 34805 Da) showed moderate homology to those of known pectate lyases in the polysaccharide lyase family 1. The purified recombinant enzyme had an isoelectric point of pH 9.7 and a molecular mass of 34 kDa, and exhibited a very high specific activity compared with known pectate lyases reported so far. The enzyme activity was stimulated 1.6 fold by addition of NaCl at an optimum of 100 mM. When Pel-4A was stored at 50 degrees C for 60 h, striking stabilization by 100 mM NaCl was observed in a pH range from 5 to 11.5, whereas it was stable only around pH 11 in the absence of NaCl.

Purification of alkaline proteases from a Bacillus strain and their possible interrelationship.

Alkalophilic Bacillus sp. KSM-K16 produced three alkaline proteases, as detected by polyacrylamide gel electrophoresis (PAGE). The major protease, designated M protease, was recently purified to homogeneity and its properties were characterized. In the present study, two minor proteases, designated H protease and N protease, were purified to homogeneity from cultures of this organism. H protease had a molecular mass of 28 kDa, as estimated by sodium dodecyl sulfate/PAGE (SDS-PAGE) and its maximum activity against casein was observed at pH 11.0 and at 55 degrees C. N protease consisted of two polypeptide chains with molecular masses of 12.5 kDa and 14.5 kDa, as estimated by SDS-PAGE, although it migrated as a single protein band during non-denaturing PAGE. Its maximum activity was observed at pH 11.0 and at 60 degrees C. The amino-terminal sequences of H protease and of the 14.5-kDa polypeptide of N protease were identical to that of M protease. The electrophoretic relationship between the three enzymes was examined after they had been stored at different pH values and at 5 degrees C. M protease was converted to H protease more rapidly at pH 11 than at pH 8 or below, and H protease was converted to M protease at pH 8 or below but not at pH 11. N protease appeared to be the autolytic product of the M and H proteases.

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.

Thermostabilization by replacement of specific residues with lysine in a Bacillus alkaline cellulase: building a structural model and implications of newly formed double intrahelical salt bridges.

An alkaline, mesophilic endo-1,4-beta-glucanase from alkaliphilic Bacillus sp. strain KSM-64 was significantly thermostabilized by replacement of both Asn179 and Asp194 with lysine by site-directed mutagenesis. Structural remodeling of the mutant enzyme newly generated by the double mutation suggested that Glu175-->Lys179 and Glu190-->Lys194 were the most plausible ion pairs, both of which involved side chains at the i and i + 4 positions on the alpha(4)-helix from Glu175 to Ser195. By molecular dynamics simulations, the N(zeta) hydrogens of Lys179 and Lys194 were found to coordinate with the carbonyl O(varepsilon1) and O(varepsilon2) of Glu175 and the carbonyl O(varepsilon1) of Glu190, respectively, with distances of around 2 A for all. These results confirm that the formation of these double intrahelical ion pairs (salt bridges) is responsible for the thermostabilization by the double mutation.

Thermostable alkaline cellulase from an alkaliphilic isolate, Bacillus sp. KSM-S237.

Thermostable alkaline cellulase (endo-1,4-beta-glucanase, EC 3.2.1.4) activity was detected in the culture medium of a strictly alkaliphilic strain of Bacillus, designated KSM-S237. This novel enzyme was purified to homogeneity by a two-step column-chromatographic procedure with high yield. The N-terminal amino acid sequence of the purified enzyme was Glu-Gly-Asn-Thr-Arg-Glu-Asp-Asn-Phe-Lys-His-Leu-Leu-Gly-Asn-Asp-Asn-Val- Lys-Arg. The enzyme had a molecular mass of approximately 86 kDa and an isoelectric point of pH 3.8. The enzyme had a pH optimum of 8.6-9.0 and displayed maximum activity at 45 degrees C. The alkaline enzyme was stable up to 50 degrees C and more than 30% of the original activity was detectable after heating at 100 degrees C and at pH 9.0 for 10 min. The enzyme hydrolyzed carboxymethylcellulose, lichenan (beta-1,3;1,4-linkage), and p-nitrophenyl derivatives of cellotriose and cellotetraose. Crystalline forms of cellulose (Avicel and filter paper), H3PO4-swollen cellulose, NaOH-swollen cellulose, curdlan (beta-1,3-linkage), laminarin (beta-1,3;1,6-linkage), and xylan were barely hydrolyzed at all.

Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16.

Alkaline protease (EC 3.4.21.14) activity, suitable for use in detergents, was detected in the alkaline culture medium of Bacillus sp. KSM-K16, which was originally isolated from soil. The enzyme, designated M protease, was purified to homogeneity from the culture broth by column chromatographies. The N-terminal amino acid sequence was Ala-Gln-Ser-Val-Pro-Trp-Gly-Ile-Ser-Arg- Val-Gln-Ala-Pro-Ala-Ala-His-Asn-Arg-Gly-Leu-Thr-Gly. The molecular mass of the protease was 28 kDa, and its isoelectric point was close to pH 10.6. Maximum activity toward casein was observed at 55 degrees C and at pH 12.3 in 50 mM phosphate/NaOH buffer. The activity was inhibited by phenylmethylsulfonyl fluoride and chymostatin. The enzyme was very stable in long-term incubation with liquid detergents at 40 degrees C. The enzyme cleaved the oxidized insulin B chain initially at Leu15-Tyr16 and efficiently at ten more sites. Among various oligopeptidyl p-nitro-anilides (pNA) tested, N-succinyl-Ala-Ala-Pro-Phe-pNA was efficiently hydrolyzed by M protease. M protease was precipitated in (NH4)2SO4-saturated acetate buffer (pH 5.0) as plank-like crystals.

Pancreatic arteriovenous malformation observed to bleed from the bile duct and a duodenal ulcer: report of a case.

A 48-year-old man with recurrent episodes of biliary colic and subsequent pancreatitis was admitted to undergo a cholecystectomy. A gastroduodenal fiberscopic examination was performed because of massive melena on the seventh day after admission. It revealed a shallow ulcer on the posterior wall of a duodenal bulbus with rubor and an exposed vessel, which was clipped endoscopically to stop the bleeding. Further observations showed the papilla of Vater to be bleeding from the papilla. A contrast-enhanced abdominal computed tomography scan demonstrated a dilatation of the common bile duct and several dilated vasculatures around the portal vein, some of which drained into the portal vein. Based on the angiography findings, a diagnosis of arteriovenous malformation in the pancreas head was obtained and an embolization of the gastroduodenal artery was performed. Although the melena subsided, he underwent a pylorus-preserving pancreatoduodenectomy to prevent the recurrence of hemorrhaging. The histopathological findings of the bile duct revealed inflammatory cell infiltration and a detachment of the epithelium, except in a small part of the bile duct. A rupture of a damaged vessel inside the bile duct was observed, which was thought to be the cause of hemobilia. Sections of the pancreatic head demonstrated an inflammatory lesion with fibrosis and saponification as well as a large degree of arteriovenous anastomosis. The patient was discharged on the 35th day after the operation following an uneventful postoperative course.

Recombinant adeno-associated virus vector-transduced vascular endothelial cells express the thrombomodulin transgene under the regulation of enhanced plasminogen activator inhibitor-1 promoter.

We were able to facilitate plasminogen activator inhibitor 1 (PAI-1) promoter activity approximately by 14-fold using an enhancer element. This enhanced PAI-1 promoter has a strong basal activity, comparable to CAG promoter activity, and has a response similar to the PAI-1 promoter with respect to TGFbeta 1 and TNFalpha stimulation. The characteristics of the enhanced PAI-1 promoter are thought to be suited to timely and tissue-specific expression of anticoagulant molecules in the vascular cells. Thus, we developed recombinant adeno-associated virus (rAAV) vectors using the enhanced PAI-1 promoter and were successful in transducing vascular endothelial cells to express the thrombomodulin transgene under the regulation of the enhanced PAI-1 promoter in vitro. Thromobomodulin transgene expression driven by the enhanced PAI-1 promoter in rAAV vector-transduced cultured endothelial cells was between 600- and 1000-fold higher than constitutive thrombomodulin gene expression in cultured human umbilical vein endothelial cells and was up-regulated by TGFbeta1 and TNFalpha stimulation which may down-regulate endogenous thrombomodulin gene expression in endothelial cells. The brain vascular endothelial cells of Mongolian gerbils could also be transduced by the same rAAV vector in vivo. Transduction of endothelial cells by rAAV vectors to express enhanced PAI-1 promoter-driven transgenes may be a useful gene therapy approach for vascular diseases.

Cloning and sequencing of a high-alkaline pectate lyase gene from an alkaliphilic Bacillus isolate.

Alkaliphilic Bacillus sp. strain KSM-P103 was found to exoproduce a high-alkaline pectate lyase (pectate transeliminase, EC 4.2.2.2). The gene for this enzyme from the alkaliphile was cloned and sequenced for the first time. The structural gene contained a 1,038-bp open reading frame encoding 345 amino acids. The deduced amino acid sequence of the mature enzyme (302 amino acids, 33,312 Da), designated Pel-103, showed very low similarity to those of known pectate lyases with 28-36% identity: the loop regions were very short and the amino acid usage in the parallel beta-helix core structure was considerably different. Moreover, physicochemical and catalytic properties of Pel-103 were different from those of other enzymes reported so far. Pel-103 was a very basic protein with an isoelectric point close to pH 10.5 and had optimal activity at 60-65 degrees C and at pH as high as 10.5. However, Pel-103 appeared to have a similar core and active site topology to the enzymes of known structure from Erwinia chrysanthemi and Bacillus subtilis. Expression of the gene for Pel-103 in B. subtilis resulted in high pectate lyase activity in the culture broth, concomitant with the appearance of a main protein band on an SDS gel at 33 kDa.

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).