Lipases (EC 3.1.1.3) are enzymes used in the oils and fats industries to modify the physicochemical properties of triacylglycerol (TAG). Lipase-catalyzed interesterification at high temperatures is an effective method for modifying the physicochemical properties of TAG. The lipase from Burkholderia plantarii (BpL) exhibits excellent catalytic activity for non-regiospecific interesterification at high temperatures, with depressed lipase hydrolytic activity. The detailed catalytic mechanism for reactions involving catalytic residues has not been elucidated because of the lack of a conventional method for estimating interesterification activity. We used our original water-in-oil emulsion system to estimate the interesterification activity of lipases. BpL showed 10% hydrolytic and 140% interesterification activities compared to the lipase from Burkholderia cepacia, which has a high sequence homology with BpL. By comparing the sequence and crystal structure data of the lipases, we clarified that two amino acids near the active center are one of the factors controlling the hydrolytic and interesterification activities of the enzyme.
The molecular pathological mechanisms underlying schizophrenia remain unclear; however, genomic analysis has identified genes encoding important risk molecules. One such molecule is neurexin 1alpha (NRXN1alpha), a presynaptic cell adhesion molecule. In addition, novel autoantibodies that target the nervous system have been found in patients with encephalitis and neurological disorders. Some of these autoantibodies inhibit synaptic antigen molecules. Studies have examined the association between schizophrenia and autoimmunity; however, the pathological data remain unclear. Here, we identified a novel autoantibody against NRXN1alpha in patients with schizophrenia (n=2.1%) in a Japanese cohort (n=387). None of the healthy control participants (n=362) were positive for anti-NRXN1alpha autoantibodies. Anti-NRXN1alpha autoantibodies isolated from patients with schizophrenia inhibited the molecular interaction between NRXN1alpha and Neuroligin 1 (NLGN1) and between NRXN1alpha and Neuroligin 2 (NLGN2). Additionally, these autoantibodies reduced the frequency of the miniature excitatory postsynaptic current in the frontal cortex of mice. Administration of anti-NRXN1alpha autoantibodies from patients with schizophrenia into the cerebrospinal fluid of mice reduced the number of spines/synapses in the frontal cortex and induced schizophrenia-related behaviors such as reduced cognition, impaired pre-pulse inhibition, and reduced social novelty preference. These changes were improved through the removal of anti-NRXN1alpha autoantibodies from the IgG fraction of patients with schizophrenia. These findings demonstrate that anti-NRXN1alpha autoantibodies transferred from patients with schizophrenia cause schizophrenia-related pathology in mice. Removal of anti-NRXN1alpha autoantibodies may be a therapeutic target for a subgroup of patients who are positive for these autoantibodies.
An 86-year-old woman intentionally drank approximately 300 mL of a glyphosate-surfactant. She was found with consciousness disturbance and experienced several vomiting episodes. On arrival, serum biochemistry revealed a decreased level of butyrylcholinesterase (B-CHE) [11 (normal range: 180-450) IU/L]. Later, her B-CHE level further decreased to single-digit values, and she became comatose with involuntary movement and an increase in muscle tone. Her consciousness level and muscle tone improved with the recovery of her B-CHE level. Physicians should be alert for the occurrence of intermediate syndrome when the B-CHE levels of patients who have consumed a massive amount of glyphosate-surfactant show a prolonged decrease.
Title: Crystal structure of an acetylesterase from Talaromyces cellulolyticus and the importance of a disulfide bond near the active site Watanabe M, Fukada H, Inoue H, Ishikawa K Ref: FEBS Letters, 589:1200, 2015 : PubMed
Carbohydrate esterase catalyzes the de-O or de-N-acylation of substituted saccharides in plant cell walls and thus has great potential for industrial biomass saccharification. We recently identified the putative carbohydrate esterase family 3 (CE3) from Talaromyces cellulolyticus. Here, we prepared the recombinant catalytic domain of the enzyme and crystallized it. The crystal structure was determined to 1.5A resolution. From the structural analysis, it was elucidated that a n-octyl-beta-d-glucopyranoside bound to near the catalytic triad (Ser10, Asp179 and His182) and was buried in the active site cavity. Site-directed mutagenesis showed that the N-terminal disulfide bond located near the catalytic triad is involved in the activity and structural stability of the enzyme.
A feruloyl esterase catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from esterified sugars in plant cell walls. Talaromyces cellulolyticus is a high cellulolytic-enzyme producing fungus. However, there is no report for feruloyl esterase activity of T. cellulolyticus. Analysis of the genome database of T. cellulolyticus identified a gene encoding a putative feruloyl esterase B. The recombinant enzyme was prepared using a T. cellulolyticus homologous expression system and characterized. The purified enzyme exhibited hydrolytic activity toward p-nitrophenyl acetate, p-nitrophenyl trans-ferulate, methyl ferulate, rice husk, and bagasse. HPLC assays showed that the enzyme released ferulic acid and p-coumaric acid from hydrothermal-treated rice husk and bagasse. Trichoderma sp. is well-known high cellulolytic-enzyme producing fungus useful for the lignocellulosic biomass saccharification. Interestingly, no feruloyl esterase has been reported from Trichoderma sp. The results show that this enzyme is expected to be industrially useful for biomass saccharification.
Title: Effects of donepezil dose escalation in Parkinson's patients with dementia receiving long-term donepezil treatment: an exploratory study Ishikawa K, Motoi Y, Mizuno Y, Kubo S, Hattori N Ref: Psychogeriatrics, 14:93, 2014 : PubMed
BACKGROUND: The benefits of escalating the dose of donepezil in patients who are already receiving long-term treatment with it have not been well evaluated. Therefore, an exploratory study to assess the effects of donepezil dose escalation in patients with Parkinson's disease with dementia, and specifically on patients receiving long-term treatment with donepezil, was performed. METHODS: Patients treated with 5-mg/day donepezil for at least 3 months and having a Mini-Mental State Examination (MMSE) score between 10 and 26 were included in this study. Donepezil dosage was then increased to 10 mg/day for 12 weeks. The outcome measures were a modified form of the Neuropsychiatric Inventory (NPI) with an extra domain for additional evaluation of fluctuation in cognitive functions (NPI-11) and the MMSE. RESULTS: Of the nine patients enrolled, two withdrew because of nausea and inability to be assessed on the predetermined date; this left seven participants (four men and three women) with a mean age of 74.6 +/- 6.9 years, a mean period of Parkinson's disease of 11.7 +/- 7.5 years, and median donepezil use of 7 months (range: 3-56 months). At baseline, the mean total NPI-11 and mean MMSE scores were 18.3 +/- 5.6 points and 21.3 +/- 5.3 points, respectively. At week 12, they improved by 8.3 points (P < 0.01) and 3.0 points (P = 0.08), respectively, from the baseline. The NPI symptom domains that improved by 1 or more points were hallucination (1.3 points), depression (1.0 points), anxiety (1.6 points), and aberrant motor behaviour (1.7 points). None of the patients withdrew because of worsening of parkinsonism. CONCLUSIONS: The present results suggest that treatment with dose escalation of donepezil from 5 mg/day to 10 mg/day may be therapeutically useful for patients with Parkinson's disease with dementia who have taken donepezil 5 mg/day in the long term.
Title: Crystallization and preliminary X-ray crystallographic analysis of a putative feruloyl esterase from Talaromyces cellulolyticus Watanabe M, Ishikawa K Ref: Acta Crystallographica F Struct Biol Commun, 70:1664, 2014 : PubMed
Feruloyl esterase (FAE; EC 3.1.1.73) catalyzes the cleavage of the ester bond between ferulic acid and polysaccharides in plant cell walls, and thus holds significant potential for the industrial utilization of biomass saccharification. A feruloyl esterase was identified from the genome database of Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus). The gene consists of the catalytic domain and a carbohydrate-binding module connected through a serine/threonine-rich linker region. The recombinant enzyme was prepared, purified and crystallized at 293 K using 0.1 M imidazole pH 8.0, 0.2 M calcium acetate, 14% PEG 8000 as the precipitant. The crystal diffracted to 2.6 A resolution and the crystal system is primitive orthorhombic, with unit-cell parameters a = 90.9, b = 123.4, c = 135.4 A. Four molecules are assumed to be present per asymmetric unit, corresponding to a Matthews coefficient of 2.50 A(3) Da(-1) and a solvent content of 50.88%(v/v).
Title: Vasculitis, Atherosclerosis, and Altered HDL Composition in Heme-Oxygenase-1-Knockout Mice Ishikawa K, Navab M, Lusis AJ Ref: Int J Hypertens, 2012:948203, 2012 : PubMed
To elucidate roles of heme oxygenase-1 (HO-1) in cardiovascular system, we have analyzed one-year-old HO-1-knockout mice. Homozygous HO-1-knockout mice had severe aortitis and coronary arteritis with mononuclear cellular infiltration and fatty streak formation even on a standard chow diet. Levels of plasma total cholesterol and HDL were similar among the three genotypes. However, homozygous HO-1-knockout mice had lower body weight and plasma triglyceride. HO-1-deficiency resulted in alteration of the composition of HDL. The ratio of apolipoprotein AI to AII in HO-1-knockout mice was reduced about 10-fold as compared to wild-type mice. In addition, paraoxonase, an enzyme against oxidative stress, was reduced less than 50% in HO-1-knockout mice. The knockout mice also exhibited significant elevation of plasma lipid hydroperoxides. This study using aged HO-1-knockout mice strengthened the idea that HO-1 functions to suppress systemic inflammation in artery wall and prevents plasma lipid peroxidation.
Here, we report a case of rapid eye movement sleep behavioural disorder in an elderly patient with dementia with Lewy bodies. Pretreatment polysomnography revealed atonia during rapid eye movement sleep, absence of sleep spindles and loss of slow-wave sleep. Administration of donepezil, an acetylcholinesterase inhibitor, markedly improved delusional symptoms and cognitive function. Pretreatment polysomnography performed after donepezil administration revealed a considerable number of sleep spindles. The effects of cholinergic modulation induced by donepezil seemed to cause remarkable improvement in mental status, incorporating associated with sleep spindles generated by the thalamocortical circuit involved in this patient.
BACKGROUND: Downbeat nystagmus (DBN) is often seen in patients with pure cerebellar type of spinocerebellar ataxia (SCA) like spinocerebellar ataxia type 6 (SCA6). DBN frequently presents with other cerebellar symptoms such as postural imbalance or ataxia. A potassium channel blocker 3,4-diaminopyridine (3,4-DAP) has been reported to reduce DBN by increasing the excitability of Purkinje cells. OBJECTIVE: The objective of this study is to determine whether 3,4-DAP has a beneficial effect on DBN along with postural imbalance and ataxic symptoms in 10 patients with SCA6 and five patients with chromosome 16q22.1-linked autosomal dominant cerebellar ataxia (16q-ADCA). RESULTS: The patients took 20mg of 3,4-DAP twice a day for a week. DBN was observed in seven patients with SCA6 and two with 16q-ADCA. Although 3,4-DAP significantly reduced DBN (P<0.05), other ataxic symptoms did not improved. However, 3,4-DAP showed benefit in two patients with oscillopsia. CONCLUSION: 3,4-DAP may be effective on DBN and oscillopsia, although it was not proved to be effective on other symptoms of ataxia in SCA patients.
From postnatal-day-0 to postnatal-day-2, a few acetylcholinesterase (AChE)-active and choline acetytransferase (ChAT)-immunoreactive nerve fibers and relatively numerous vesicular acetylcholine transporter (VAChT)-immunoreactive puncta were observed in the rat adrenal medulla. Despite relatively numerous clear vesicles in the nerve fibers, the synthesis and hydrolysis of acetylcholine may not be fully activated until postnatal-day-2. The number of AChE-active and ChAT-immunoreactive nerve fibers dramatically increased and that of VAChT-immunoreactive puncta gradually increased from postnatal-day-3 to postnatal-week-1. The synthesis and hydrolysis of acetylcholine may be dramatically activated in the nerve fibers of the medulla until postnatal-week-1. From postnatal-week-2 to postnatal-week-3, the number of AChE-active and the ChAT-immunoreactive nerve fibers gradually increased and reached the adult levels. The VAChT-immunoreactive puncta per unit area was maximum number at postnatal-week-2. The synthesis and hydrolysis of acetylcholine in the nerve fibers of the medulla may be completed between postnatal-week-2 to postnatal-week-3. The diameter of the VAChT-immunoreactive puncta gradually increased from postnatal-day-0 with aging. However, the number of the VAChT-immunoreactive puncta gradually decreased from postnatal-week-2 onwards. In electron-microscopy, the VAChT-immunoreactive deposits were seen in clusters of clear vesicles, and the diameter of the nerve fibers and the number of clear vesicles at postnatal-week-8 increased compared with those at postnatal-week-2. The AChE-active, ChAT-immunoreactive, and VAChT-immunoreactive nerve fibers observed around noradrenaline (NA) cells were denser than those around adrenaline (A) cells in the medulla at postnatal-week-8. These suggest that the preferential innervation of NA and A cells may cause the differential secretion NA and A.
Title: Irinotecan therapy in a 12-year-old girl with recurrent brain stem glioma and without functional polymorphisms in UGT1A1 activity: case report Ishikawa K, Kajita Y, Hasegawa Y, Noda Y, Yoshida J, Nabeshima T Ref: J Neurooncol, 74:283, 2005 : PubMed
A 10-year-old girl was diagnosed with astrocytoma grade 2. Immuno-chemo-radiotherapy (interferon, ranimustine, and radiation), second-line chemotherapy (carboplatin and etoposide, 7 cycles) and third-line chemotherapy (ifosfamide, carboplatin, and etoposide) was given to treat progressive disease. Finally, irinotecan therapy was initiated and led to dramatic clinical improvement. Irinotecan is metabolized by carboxylesterase to form an active SN-38, which is further conjugated and detoxified by UDP-glucuronosyltransferase (UGT) to yield its beta-glucuronide. The polymorphic UGT isoenzyme, UGT1A1 has genetic variants which decrease in SN-38 glucuronidating capacity and could help predict irinotecan-associated toxicity. The patient suffered excessive toxicity with low-dose irinotecan although no functional polymorphism in UGT1A1 was identified. We suggest that irinotecan offers an effective treatment option for children with recurrent brain stem glioma and other genetic variants except UGT1A1 may be a risk factor for irinotecan-induced toxicity.
Title: Prediction of the coding sequences of unidentified human genes. XVII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro Nagase T, Kikuno R, Ishikawa K, Hirosawa M, Ohara O Ref: DNA Research, 7:143, 2000 : PubMed
To provide information regarding the coding sequences of unidentified human genes, we have conducted a sequencing project of human cDNAs which encode large proteins. We herein present the entire sequences of 100 cDNA clones of unknown human genes, named KIAA1444 to KIAA1543, from two sets of size-fractionated human adult and fetal brain cDNA libraries. The average sizes of the inserts and corresponding open reading frames of cDNA clones analyzed here were 4.4 kb and 2.6 kb (856 amino acid residues), respectively. Database searches of the predicted amino acid sequences classified 53 predicted gene products into the following five functional categories: cell signaling/communication, nucleic acid management, cell structure/motility, protein management and metabolism. It was also revealed that homologues for 32 KIAA gene products were detected in the databases, which were similar in sequence through almost their entire regions. Additionally, the chromosomal loci of the genes were determined by using human-rodent hybrid panels unless their chromosomal loci were already assigned in the public databases. The expression levels of the genes were monitored in spinal cord, fetal brain and fetal liver, as well as in 10 human tissues and 8 brain regions, by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.
We have conducted a sequencing project of human cDNAs which encode large proteins in brain. For selection of cDNA clones to be sequenced in this project, cDNA clones have been experimentally examined by in vitro transcription/translation prior to sequencing. In this study, we tested an alternative approach for picking up cDNA clones having a high probability of carrying protein coding region. This approach exploited 5'-end single-pass sequence data and the GeneMark program for assessing protein-coding potential, and allowed us to select 74 clones out of 14,804 redundant cDNA clones. The complete sequence data of these 74 clones revealed that 45% of them encoded proteins consisting of more than 500 amino acid residues while all the clones thus selected carried possible protein coding sequences as expected. The results indicated that the GeneMark analysis of 5'-end sequences of cDNAs offered us a simple and effective means to select cDNA clones with protein-coding potential although the sizes of the encoded proteins could not be predicted.
To extend our cDNA project for accumulating basic information on unidentified human genes, we newly determined the sequences of 100 cDNA clones from a set of size-fractionated human adult and fetal brain cDNA libraries, and predicted the coding sequences of the corresponding genes, named KIAA1019 to KIAA1118. The sequencing of these clones revealed that the average size of the inserts and corresponding open reading frames were 5.0 kb and 2.6 kb (880 amino acid residues), respectively. Database search of the predicted amino acid sequences classified 58 predicted gene products into the five functional categories, such as cell signaling/communication, cell structure/motility, nucleic acid management, protein management and cell division. It was also found that, for 34 gene products, homologues were detected in the databases, which were similar in sequence through almost the entire regions. The chromosomal locations of the genes were determined by using human-rodent hybrid panels unless their mapping data were already available in the public databases. The expression profiles of all the genes among 10 human tissues, 8 brain regions (amygdala, corpus callosum, cerebellum, caudate nucleus, hippocampus, substania nigra, subthalamic nucleus, and thalamus), spinal cord, fetal brain and fetal liver were also examined by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.
Title: Prediction of the coding sequences of unidentified human genes. XV. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro Nagase T, Ishikawa K, Kikuno R, Hirosawa M, Nomura N, Ohara O Ref: DNA Research, 6:337, 1999 : PubMed
In order to obtain information on the coding sequences of unidentified human genes, we newly determined the sequences of 100 cDNA clones of unknown human genes, which we named KIAA1193 to KIAA1292, from two sets of size-fractionated human adult and fetal brain cDNA libraries. The results of our particular strategy to select cDNA clones which have the potentiality of coding for large proteins in vitro revealed that the average sizes of the inserts and the corresponding open reading frames reached 5.2 kb and 2.8 kb (933 amino acid residues), respectively. By the computational analysis of the predicted amino acid sequences against the OWL and Pfam databases, 58 predicted gene products were classified into the following five functional categories: cell signaling/communication, cell structure/motility, nucleic acid management, protein management and metabolism. It was also found that 30 gene products had homologues in the public databases which were similar in sequence throughout almost their entire regions to the newly identified genes. The chromosomal loci of the genes were assigned by using human-rodent hybrid panels unless their mapping data were already available in the public databases. The expression profiles of the genes were studied in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.
As a part of our cDNA project for deducing the coding sequence of unidentified human genes, we newly determined the sequences of 100 cDNA clones from a set of size-fractionated human brain cDNA libraries, and predicted the coding sequences of the corresponding genes, named KIAA0919 to KIAA1018. The sequencing of these clones revealed that the average sizes of the inserts and corresponding open reading frames were 4.9 kb and 2.6 kb (882 amino acid residues), respectively. A computer search of the sequences against the public databases indicated that predicted coding sequences of 87 genes contained sequences similar to known genes, 53% of which (46 genes) were categorized as proteins relating to cell signaling/communication, cell structure/motility and nucleic acid management. The chromosomal locations of the genes were determined by using human-rodent hybrid panels unless their mapping data were already available in the public databases. The expression profiles of all the genes among 10 human tissues, 8 brain regions (amygdala, corpus callosum, cerebellum, caudate nucleus, hippocampus, substania nigra, subthalamic nucleus, and thalamus), spinal cord, fetal brain and fetal liver were also examined by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.
When the genome of the thermophilic archaeon Pyrococcus horikoshii was sequenced, a gene homologous to the mammalian gene for an acylamino acid-releasing enzyme (EC 3.4.19.1) was found in which the enzyme's proposed active residues were conserved. The P. horikoshii gene comprised an open reading frame of 1,896 base pairs with an ATG initiation codon and a TAG termination codon, encoding a 72,390-Da protein of 632 amino acid residues. This gene was overexpressed in Escherichia coli with the pET vector system, and the resulting enzyme showed the anticipated amino-terminal sequence and high hydrolytic activity for acylpeptides. This enzyme was concluded to be the first acylamino acid-releasing enzyme from an organism other than a eukaryotic cell. The existence of the enzyme in archaea suggests that the mechanisms of protein degradation or initiation of protein synthesis or both in archaea may be similar to those in eukaryotes. The enzyme was stable at 90 degreesC, with its optimum temperature over 90 degreesC. The specific activity of the enzyme increased 7-14-fold with heat treatment, suggesting the modification of the enzyme's structure for optimal hydrolytic activity by heating. This enzyme is expected to be useful for the removal of Nalpha-acylated residues in short peptide sequence analysis at high temperatures.
Title: Prediction of the coding sequences of unidentified human genes. X. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro Ishikawa K, Nagase T, Suyama M, Miyajima N, Tanaka A, Kotani H, Nomura N, Ohara O Ref: DNA Research, 5:169, 1998 : PubMed
As an extension of our cDNA analysis for deducing the coding sequences of unidentified human genes, we have newly determined the sequences of 100 cDNA clones from a set of size-fractionated human brain cDNA libraries, and predicted the coding sequences of the corresponding genes, named KIAA0611 to KIAA0710. In vitro transcription-coupled translation assay was applied as the first screening to select cDNA clones which produce proteins with apparent molecular mass of 50 kDa and over. One hundred unidentified cDNA clones thus selected were then subjected to sequencing of entire inserts. The average size of the inserts and corresponding open reading frames was 4.9 kb and 2.8 kb (922 amino acid residues), respectively. Computer search of the sequences against the public databases indicated that predicted coding sequences of 87 genes were similar to those of known genes, 62% of which (54 genes) were categorized as proteins related to cell signaling/communication, cell structure/motility and nucleic acid management. The expression profiles in 10 human tissues of all the clones characterized in this study were examined by reverse transcription-coupled polymerase chain reaction and the chromosomal locations of the clones were determined by using human-rodent hybrid panels.
As a part of our project for accumulating sequence information of the coding regions of unidentified human genes, we herein report the sequence features of 78 new cDNA clones isolated from human brain cDNA libraries as those which may code for large proteins. The sequence data showed that the average size of the cDNA inserts and their open reading frames was 6.0 kb and 2.8 kb (925 amino acid residues), respectively, and these clones produced the corresponding sizes of protein products in an in vitro transcription/translation system. Homology search against the public databases indicated that the predicted coding sequences of 68 genes contained sequences similar to known genes, 69% of which (47 genes) were related to cell signaling/communication, nucleic acid management, and cell structure/motility. The expression profiles of these genes in 14 different tissues have been analyzed by the reverse transcription-coupled polymerase chain reaction method, and 8 genes were found to be predominantly expressed in the brain.
Title: Cloning and sequencing of the gene for type I carboxypeptidase in rice Washio K, Ishikawa K Ref: Biochimica & Biophysica Acta, 1199:311, 1994 : PubMed
Title: Organ-specific and hormone-dependent expression of genes for serine carboxypeptidases during development and following germination of rice grains Washio K, Ishikawa K Ref: Plant Physiol, 105:1275, 1994 : PubMed
Several cDNA clones encoding either serine carboxypeptidases or related proteins of Oryza sativa L. were identified, and the abundance of the corresponding mRNA in immature and germinated grains was examined. The deduced amino acid sequence of each cDNA included key sequences, such as a pentapeptide (G-X-S-X-G/A) that is conserved among many serine carboxypeptidases, and the putative protein products were classified as two general and one novel type of cereal serine carboxypeptidases. Two general types exhibited considerable homology to type I and type III carboxypeptidases of cereal plants. The novel type encoded a serine carboxypeptidase-like protein that was very similar to type III carboxypeptidases of barley and wheat but had slight differences in both the N- and the C-terminal sequences. The mRNAs of each of these carboxypeptidases were observed in immature grains, and they decreased during maturation. The abundance of mRNA for each class of carboxypeptidase increased again following germination with the same time course and in a tissue-specific manner. The mRNAs for type I and type III-like carboxypeptidases were abundant in germinated embryos composed of leaf, root, and scutellum, whereas the mRNA for type III carboxypeptidase was conspicuous in endosperm that contained the aleurone layer. Altered amounts of mRNA in deembryonated half-grains in response to phytohormones, such as gibberellic acid and abscisic acid, were only detectable in the case of type III carboxypeptidase. Southern blot analysis using rice genomic DNA revealed the simple organization of each gene for these three classes of carboxypeptidases.
Title: Structure and expression during the germination of rice seeds of the gene for a carboxypeptidase Washio K, Ishikawa K Ref: Plant Mol Biol, 19:631, 1992 : PubMed
An in vitro comparison demonstrated that the concentration of NIK-247 that inhibited cholinesterase (ChE) activities to half the normal level (ID50) was 1.3 x 10(-6) M. This value was higher than those for both physostigmine (PHY; 1.2 x 10(-7) M) and tetrahydroaminoacridine (THA; 3.6 x 10(-7) M), which are used as cholinesterase inhibitors in the treatment of cholinergic deficits. Neither NIK-247 nor THA affected the activity of choline acetyltransferase (ChAT). These inhibitions of ChE by NIK-247 and PHY lasted for 2 h, while that by THA lasted for over 4 h. In the effects of NIK-247 and PHY, the concentrations of intrastriatal acetylcholine (ACh) were changed in relation to the inhibition of the ChE activity. However, THA caused a transient increase in the ACh level lasting for only 2 h instead of inhibiting the enzyme activity for over 4 h. These findings suggest that NIK-247 is a drug with a similar profile in its effect on cholinergic neurons to PHY, the prototype drug among ChE inhibitors. The data indicate that NIK-247 may be useful as a drug for the treatment of central as well as peripheral deficits of the cholinergic mechanism.
The extracellular poly(3-hydroxybutyrate) depolymerase gene from Alcaligenes faecalis T1 was cloned into Escherichia coli DH1 by using the plasmid pUC8. An A. faecalis T1 genomic library was prepared in E. coli from a partial Sau3AI digest and screened with antibody against the depolymerase. Of the 29 antibody-positive clones, 1 (pDP14), containing about 4 kilobase pairs of A. faecalis T1 DNA, caused expression of a high level of depolymerase activity in E. coli. The enzyme purified from E. coli was not significantly different from the depolymerase of A. faecalis in molecular weight, immunological properties, peptide map, specific activity, or substrate specificity. Most of the expressed enzyme was found to be localized in the periplasmic space of E. coli, although about 10% of the total activity was found in the culture medium. Results of a deletion experiment with pDP14 showed that a large SalI fragment of about 2 kilobase pairs was responsible for expression of the enzyme in E. coli. The nucleotide sequence of the large SalI fragment has been determined. Comparison of the deduced amino terminus with that obtained from sequence analysis of the purified protein indicated that poly(3-hydroxybutyrate) depolymerase exists as a 488-amino-acid precursor with a signal peptide of 27 amino acids.
