An anatomic study of the accessory anterolateral talar facet.

BACKGROUND: A small accessory facet with articular surface morphology is occasionally seen on the talus, bordering on the lateral end of the sinus tarsi. This facet has been named the accessory anterolateral talar facet. However, few anatomical studies have addressed this facet. Here we present the precise morphology of accessory anterolateral talar facet with emphasis on anatomical correlation between the presence of this facet and the angle of the infero-lateral surface of the talus (talar infero-lateral surface - TILS angle). MATERIALS AND METHODS: A total of 22 (11 male, 11 female) adult cadavers with no known pathological conditions in the talocalcaneal joints were examined during educational dissection at Nagoya City University Medical School in 2013. After exclusion of 1 joint due to the poor condition of the talus, 43 talus (22 right, 21 left) were analysed. We judged the presence of the accessory anterolateral talar facet and measured TILS angle. We performed statistical analysis on the point of laterality, gender difference and the difference in the TILS angles in tali with or without the accessory anterolateral talar facets. RESULTS: An accessory anterolateral talar facet was identified in 11 (26%) of the 43 specimens. Of the 21 cadavers with paired talar specimens, 5 displayed the facet bilaterally. CONCLUSIONS: There was no sex difference and no significant laterality, however we found that TILS angle was significantly larger in accessory anterolateral talar facet positive samples than in negative ones.

Thiol-disulfide exchange between nuclear-encoded and chloroplast-encoded subunits of pea acetyl-CoA carboxylase.

Fatty acid synthesis in pea chloroplasts is regulated by light/dark. The regulatory enzyme acetyl-CoA carboxylase is modulated by light/dark, presumably under redox regulation. Acetyl-CoA carboxylase is a multienzyme complex composed of biotin carboxylase and carboxyltransferase (CT). To demonstrate the redox regulation of CT, composed of the nuclear-encoded alpha and the chloroplast-encoded beta subunits, we identified the cysteine residues involved in such regulation. We expressed the recombinant CT in Escherichia coli and found that the partly deleted CT was, like the full-length CT, sensitive to a redox state. Site-directed mutagenesis of the deleted CT showed that replacement by alanine of the cysteine residue 267 in the alpha polypeptide or 442 in the beta polypeptide resulted in redox-insensitive CT and broke the intermolecular disulfide bond between the alpha and beta polypeptides. Similar results were confirmed in the full-length CT. These results indicate that the two cysteines in recombinant CT are involved in redox regulation by intermolecular disulfide-dithiol exchange between the alpha and beta subunits. Immunoblots of extract from plants incubated in the light or dark supported that such a disulfide-dithiol exchange is relevant in vivo. A covalent bond between a nuclear-encoded polypeptide and a chloroplast-encoded polypeptide probably regulates the enzyme activity in response to light.

Chloroplast RNA editing required for functional acetyl-CoA carboxylase in plants.

RNA editing is an important post-transcriptional process in chloroplasts and is thought to be functionally significant. Here we show a requirement of RNA editing for a functional enzyme. In peas, acetyl-CoA carboxylase (ACCase), a key enzyme of fatty acid synthesis, is composed of biotin carboxylase with the biotin carboxyl carrier protein and carboxyltransferase (CT). CT is composed of the nuclear-encoded alpha polypeptide and the chloroplast-encoded beta polypeptide in peas. One nucleotide of the beta polypeptide mRNA, which is edited in pea chloroplasts, converts the serine codon to the leucine codon. We show that this RNA editing is required for functional CT by comparing the unedited and edited recombinant enzymes. In plants not having a leucine codon at the same position, editing was shown to take place so as to create the leucine codon, indicating that editing is needed for in vivo CT activity and therefore for ACCase. To our knowledge, ACCase is an essential enzyme, suggesting that the chloroplast RNA editing is necessary for these plants.

Synthesis of carbocyclic nucleosides as potential antiviral agents.

Treatment of 1-[(1'R,2'S,3'S,4'S)-4'-methanesulfonyl-2',3'-O- isopropylidenedioxycyclopentan-1'-yl]-1-H-uracil with LiN3 gave 1-[(1'R,2'S,3'R)-2',3'-O-isopropylidenedioxy-4'- cyclopenten-1'-yl]-1-H-uracil. When KOBut was added instead of LiN3, 1-[(1'R,2'S)-2',3'-O- isopropylidenedioxy-3'-cyclopenten-1'-yl]-1-H-uracil was produced. An analogous treatment of the methanesulfonyl derivative of thymine was also carried out.

Recombinant carboxyltransferase responsive to redox of pea plastidic acetyl-CoA carboxylase.

Acetyl-CoA carboxylase regulates the rate of fatty acid synthesis. This enzyme in plants is localized in plastids and is believed to be composed of biotin carboxyl carrier protein, biotin carboxylase, and carboxyltransferase made up of alpha and beta polypeptides, although the enzyme has not been purified yet. Accumulated evidence shows that pea plastidic acetyl-CoA carboxylase is activated by light and the activation is caused by light-dependent reduction of carboxyltransferase, but not of biotin carboxylase, via a redox cascade. To understand the reductive activation of carboxyltransferase at the molecular level here, we obtained the active enzyme composed of decahistidine-tagged (His tag) alpha and beta polypeptides through the expression of the pea plastidic carboxyltransferase gene in Escherichia coli. Gel filtration showed that the molecular size of the recombinant carboxyltransferase is in agreement with that of partially purified carboxyltransferase from pea chloroplasts. The catalytic activity of the recombinant enzyme was similar to that of native carboxyltransferase. These results indicate that the molecular structure and conformation of recombinant carboxyltransferase resemble those of its native counterpart and that native carboxyltransferase is indeed composed of alpha and beta polypeptides. This recombinant enzyme was activated by dithiothreitol, a known reductant of S-S bonds, with a profile similar to that of its native counterpart. The recombinant enzyme was activated by reduced thioredoxin-f, a signal transducer of redox potential in chloroplasts under irradiation. Thus, this enzyme was redox-regulated, like that of the native carboxyltransferase.

Molecular cloning and characterization of ATP-phosphoribosyl transferase from Arabidopsis, a key enzyme in the histidine biosynthetic pathway.

We have characterized two isoforms of ATP-phosphoribosyl transferase (ATP-PRT) from Arabidopsis (AtATP-PRT1 [accession no. AB025251] and AtATP-PRT2), catalyzing the first step of the pathway of hisidine (His) biosynthesis. The primary structures deduced from AtATP-PRT1 and AtATP-PRT2 cDNAs share an overall amino acid identity of 74.6% and contain N-terminal chloroplast transit peptide sequences. DNA-blot analyses indicated that the ATP-PRTs in Arabidopsis are encoded by two separate genes with a closely similar gene structural organization. Both gene transcripts were detected throughout development, and protein-blot analysis revealed predominant accumulation of the AtATP-PRT proteins in Arabidopsis leaves. The His auxotrophy of a his1 mutant of Saccharomyces cerevisiae was suppressed by the transformation with AtATP-PRT1 and AtATP-PRT2 cDNAs, indicating that both isoforms are functionally active ATP-PRT enzymes. The K(m) values for ATP and phosphoribosyl pyrophosphate of the recombinant AtATP-PRT proteins were comparable to those of the native ATP-PRTs from higher plants and bacteria. It was demonstrated that the recombinant AtATP-PRTs were inhibited by L-His (50% inhibition of initial activity = 40-320 microM), suggesting that His biosynthesis was regulated in plants through feedback inhibition by L-His.

Synthesis of carbocyclic nucleosides and their SAH hydrolase inhibitory activities.

The cellular enzyme S-adenosyl-L-homocysteine (SAH) hydrolase has emerged as a target enzyme for the molecular design of anti-viral agents. Recently, SAH hydrolase has been considered as an attractive target in parasite chemotherapy for malaria. We report synthesis of several carbocyclic purine nucleosides and their inhibitory activities against human and malaria recombinant SAH hydrolases.

[Basic study of Beutin's washed sheep blood agar plate used for selective screening of verocytotoxin-producing/enterohemorrhagic Escherichia coli (VTEC/EHEC)].

Enterohemolysin production of Beutin's washed sheep blood agar plate has been used as an epidemiological marker for selective screening of Verocytotoxin-producing/Enterohemorrhagic Escherichia coli (VTEC/EHEC). In this study, we examined about the component of Beutin's washed sheep blood agar for further improvement of hemolysin production media. The following items were studied: the numbers of washings of sheep blood with phosphate buffered saline (PBS) (pH 7.2), concentration of sheep blood, kind and concentration of divalent metal ion (Ca2+, Mg2+) and basal medium. Twenty-seven strains of VT-producing E. coli of 7 different O-serotypes, 74 strains of VT-nonproducing E. coli of 24 different O-serotypes and one strain of O157 coded Escherichia hermanii were used for this basic study. In comparison of washing times of sheep blood with PBS, 5 times washing was better than 3 times, the original. In sheep blood concentration, supplement with 4% sheep blood was best for hemolysis observation. In experiment of addition of 2 divalent metal ions, Ca2+ and Mg2+, supplement with Ca2+ was more suitable than Mg2+ for hemolysis, and the supplement with 10 mM CaCl2, the original, was the best concentration. On the basal medium used in Beutin's sheep washed blood agar, 4 kinds of media were compared. In addition to Soybean-Casein Digest (SCD) agar, the original, Nutrient agar, Heart Infusion (HI) agar and Brain Heart Infusion (BHI) agar were examined, HI agar was the best blood agar base in the four media. As the above experimental results, the composition of the better Beutin's washed sheep blood agar may be summarize as follows: Heart Infusion agar 'Eiken' used for blood agar base, supplemented with 10 mM CaCl2 and 4% defibrinated sheep blood (Japan biosupp. Center) washed five times in phosphate-buffered saline, pH 7.2.

Light-dependent changes in redox status of the plastidic acetyl-CoA carboxylase and its regulatory component.

Plastidic acetyl-CoA carboxylase (ACCase; EC 6.4.1.2), which catalyses the synthesis of malonyl-CoA and is the regulatory enzyme of fatty acid synthesis, is activated by light, presumably under redox regulation. To obtain evidence of redox regulation in vivo, the activity of ACCase was examined in pea chloroplasts isolated from plants kept in darkness (dark-ACCase) or after exposure to light for 1 h (light-ACCase) in the presence or absence of a thiol-reducing agent, dithiothreitol (DTT). The protein level was similar for light-ACCase and dark-ACCase, but the activity of light-ACCase in the absence of DTT was approx. 3-fold that of dark-ACCase. The light-ACCase and dark-ACCase were activated approx. 2-fold and 6-fold by DTT respectively, indicating that light-ACCase was in a much more reduced, active form than the dark-ACCase. This is the first demonstration of the light-dependent reduction of ACCase in vivo. Measurement of the activities of ACCase, carboxyltransferase and biotin carboxylase in the presence and absence of DTT, and the thiol-oxidizing agent, 5, 5'-dithiobis-(2-nitrobenzoic) acid, revealed that the carboxyltransferase reaction, but not the biotin carboxylase reaction, was redox-regulated. The cysteine residue(s) responsible for redox regulation probably reside on the carboxyltransferase component. Measurement of the pH dependence of biotin carboxylase and carboxyltransferase activities in the ACCase suggested that both components affect the activity of ACCase in vivo at a physiological pH range. These results suggest that the activation of ACCase by light is caused partly by the pH-dependent activation of two components and by the reductive activation of carboxyltransferase.

[Comparison of O-serotype distribution of Escherichia coli isolated from faecal specimens of patients with sporadic diarrhea and healthy persons and regional difference in Japan].

To investigate the isolation frequency of O-serotype of Escherichia coli, a total of 1,563 faecal specimens obtained from patients with sporadic diarrhea in Ishikawa between July 1997 and June 1998, were examined. As a result of O-serotyping of isolated strains using commercially E. coli antisera (43 different types), 247 strains of 29 different O-serotypes were isolated. Isolation rate was 15.8%. Most predominant O-serotype was O1 (128 strains, 52%), followed by O18 (26 strains, 11%), O6 (17 strains, 7%), O111 (16 strains, 6%), and these 4 different O-serotypes took up three quarters of the isolated E. coli. Between August 1996 and May 1997, E. coli isolation from faecal samples of 51,893 healthy persons and O-serotyping of isolated strains using commercial antisera to 6 predominant O-serotypes (O-26, 111, 114, 128, 157 and O1) of VTEC/EHEC were carried out. Among 6 O-serotypes, the most predominant O-serotype was O1 (93% of isolates), followed by O26, 111, 128 (6%) and O114, 157 (1%). These isolation frequencies in patients were 80%, 18%, 2%, respectively, have resembled each other in healthy persons in many points. In a similar way, of these distributions of O-serotype of strains hemolysed on Beutin's blood agar plates, we compared patients with healthy persons. Fifty-six strains (3.6% of the total) of E. coli of different O-serotypes were isolated from 1,563 patients and 57 strains (2.8% of the total) belonging to 11 serotypes from 2,036 healthy persons. As a result of O-serotype frequency, both groups resembled each other. O18 and O6, the most predominant O-serotypes, occupied 64% of the isolated strains in patients and 74% in healthy persons. Next in patients, O1, 26 were 7%--level, O28 ac, 152, 157 were 4%--level, respectively, and in healthy persons. O1 was 5%--level, O28 ac, 55, 146, 152 were 4%--level respectively. In the comparison of O-serotype frequency of E. coli isolated from sporadic diarrhea in other 5 areas (Kanto district, Tokyo, Oita, Aichi and Ishikawa), O1, 6, 8, 18, 25, 26, 55, 86a, 111, 125, 126, 127a, 128, 146, 148, 157 and 166 (17 types) have covered a wide area. On the other hand, O29, 44, 78, 112ac, 115, 136, 143, 152, 168 and 169 (10 types) have a tendency to distribute in local areas, we believe that there are regional differences even in the same Japanese territory.

Synthesis of S-adenosyl-L-homocysteine hydrolase inhibitors and their biological activities.

Several nucleosides have been prepared as a possible inhibitor of human S-adenosyl-L-homocysteine (SAH) hydrolase for the development of anti-viral agents. Recently, SAH hydrolase has been considered as an attractive target for parasite chemotherapy for malaria. We report synthesis of several nucleosides including carbocyclic nucleosides and their inhibitory activities against recombinant malaria and human SAH hydrolases.

Facile method for the preparation of 7-methyl-8-oxoguanosines as an immunomodulator.

Reaction of 9-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)-7-methylguaninium iodide (2a) with hydrogen peroxide in acetic acid gave the corresponding 7-methyl-8-oxoguanosine derivative (3a) in good yield. Deprotection of 3a easily gave 7-methyl-8-oxoguanosine (1), which is well-known as an immunomodulator. Substitution of acetyl group at the N2-position of guanine ring accelerated the oxidation reaction of the 7-methylguaninium iodide.

[Selective screening of enterohemorrhagic Escherichia coli depending on hemolysis on Beutin's washed sheep blood agar plates].

Recently, Beutin et al have studied hemolysin production in a large number of serologically diverse verotoxin (VT) producing Escherichia coli (VTEC) strains and found a close association between hemolysin production and VT production. This study was to examine whether hemolysin production on sheep blood agar can be used as an epidemiological marker for microbiological screening of enterohemorrhagic Escherichia coli (EHEC). In comparison of hemolysis on washed blood agar plates prepared in our laboratory and commercially available blood agar, all VT producing strains exhibited a typical hemolytic phenotype on the former, on the other hand, VT non-producers were less hemolytic on the former than the latter. On the washed blood agar, 11 VT1 producing strains belonging to 4 different serotypes, 1 strain of VT2 producer, 5 VT1&2 producers belonging to 2 different serotypes, all the 17 strains of VTEC were positive for hemolysis. The 85 VT non-producing strains belonging to 18 different serotypes isolated from faecal specimens from patients with diarrhea were tested, 10 strains (12%) belonging to 4 different serotypes (O6, 18, 26, UT: untypable) produced hemolysin. Two-hundred four (10%) of the 2,036 strains isolated from faecal specimens from 507 healthy persons exhibited typical hemolysis. As a result of identification of these 204 hemolytic strains, 148 strains of E. coli 13 strains of Citrobacter and 10 strains of other enterobacteria, and 13 strains of Pseudomonas, 12 strains of Aeromonas, 5 strains of Proteus and 3 strains of Vibrio were detected. 148 hemolytic strains of E. coli were carried out O-serotyping with commercially available pathogenic E. coli antisera, 57 strains (39%) were classified into 11 different serotypes, but 91 strains were not typable. Most frequent serotype was O18 (24 strains), next O6 (18 strains), and other 8 different serotypes (O25, 26, 28ac, 29, 55, 146, 152, 159) were less than 2 strain respectively.

Link between light and fatty acid synthesis: thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase.

Fatty acid synthesis in chloroplasts is regulated by light. The synthesis of malonyl-CoA, which is catalyzed by acetyl-CoA carboxylase (ACCase) and is the first committed step, is modulated by light/dark. Plants have ACCase in plastids and the cytosol. To determine the possible involvement of a redox cascade in light/dark modulation of ACCase, the effect of DTT, a known reductant of S-S bonds, was examined in vitro for the partially purified ACCase from pea plant. Only the plastidic ACCase was activated by DTT. This enzyme was activated in vitro more efficiently by reduced thioredoxin, which is a transducer of redox potential during illumination, than by DTT alone. Chloroplast thioredoxin-f activated the enzyme more efficiently than thioredoxin-m. The ACCase also was activated by thioredoxin reduced enzymatically with NADPH and NADP-thioredoxin reductase. These findings suggest that the reduction of ACCase is needed for activation of the enzyme, and a redox potential generated by photosynthesis is involved in its activation through thioredoxin as for enzymes of the reductive pentose phosphate cycle. The catalytic activity of ACCase was maximum at pH 8 and 2-5 mM Mg2+, indicating that light-produced changes in stromal pH and Mg2+ concentration modulate ACCase activity. These results suggest that light directly modulates a regulatory site of plastidic prokaryotic form of ACCase via a signal transduction pathway of a redox cascade and indirectly modulates its catalytic activity via stromal pH and Mg2+ concentration. A redox cascade is likely to link between light and fatty acid synthesis, resulting in coordination of fatty acid synthesis with photosynthesis.

A Polypeptide That Induces Flowering in Lemna paucicostata at a Very Low Concentration.

A flower-inducing substance of high molecular mass, extracted from Lemna paucicostata, was purified to homogeneity. It had characteristics of a polypeptide, with an amino-terminal sequence of Leu-Val-Gly-Asn-Thr, and induced formation of flower buds of L. paucicostata 151 at a concentration of 10(-10) molar.

A flower-inducing substance of high molecular weight from higher plants.

The flower-inducing activities of aqueous extracts of several plants were fractionated by gel filtration. Three major peaks, corresponding to molecular weights of about 120, 20 to 30, and 5 to 10 kilodaltons, were detected in extracts of Lemna, Pharbitis, and Brassica. The latter two peaks may be degradation products generated during the extraction procedure. In extracts of soybean seeds, only the peak of material of 120 kilodaltons was detected. This is the first published report of a high molecular mass substance with florigenic activity in Lemna plants. The florigenic substance had some properties associated with proteins (or polypeptides), but the activity was unaffected by treatment with proteinase K.

Dose dependency of apparent volumes of distribution for methylene blue in rabbits.

The binding of methylene blue (MB) and chlordiazepoxide (CDP) to rabbit plasma was determined by ultrafiltration. The fractions (f) unbound of these two drugs to the plasma were about 23--29 ad 6--12%, respectively. These fvalues were comparable to binding data of these two drugs to bovine serum albumin. The results supported the previous suggestion stating that protein binding would partly account for relatively high value of apparent volume of distribution (Vd') for MB. Therefore, in order to investigate the distributive profile for MB as compared with that for CDP, pharmacokinetic parameters for MB and CDP were estimated from plasma concentration--time data following simultaneous intravenous administration by a bolus injection and by a contrast rate infusion in rabbits. The Vd' for MB was found to be dependent of dose in contrast with that for CDP by the infusion experiments at three different doses. These evidences assume that MB would be bound to and/or adsorbed on some biological components other than plasma protein.