Effect of Zingiber zerumbet essential oils and zerumbone inhalation on body weight of Sprague Dawley rat.

Zingiber zerumbet contained the typically essential oils. The research aims to evaluate the effect Z. zerumbet essential oil and zerumbone inhlation on rats body weight, food consumption, parasympathetic nerve activity and brown adipose tissue temperature. The essential oils of Z. zerumbet was isolated from the rhizome of Z. zerumbet. The component in the oil and zerumbone structure was determined by gas chromatography-mass spectroscopy. The structure of zerumbone crystal was determined by nuclear magnetic resonance spectroscopy. The Sprague dawley male adult rats were divided into 4 groups namely Normal Diet (ND) group, High Fat Diet (HFD) group, HFD inhaled Z. zerumbet essential oils group and HFD inhaled zerumbone group. The results showed that inhalation of Z. zerumbet essential oils and zerumbone increased the food consumption as well as increased the body weight. The increasing body weight of rats which inhaled Z. zerumbet essential oils and zerumbone is by decreasing the sympathetic nerve activity. In conclusion, inhaling Z. zerumbet essential oils and zerumbone as the major component of the oils increased the weight gain.

Successful surgical treatment of two cases of congenital chylous ascites.

The authors report on 2 patients with congenital chylous ascites who underwent successful lymphatic duct ligation after a laparoscopic lymphoid dye test. Fetal ascites had been detected in both cases, and both babies were born with marked abdominal swelling. Given that conservative treatment by medium-chain triglyceride (MCT) milk and total parenteral nutrition (TPN) was ineffective, the authors elected to perform lymphatic duct ligation on the 95th postnatal day in the former case and on the 27th postnatal day in the latter case. Lipophilic dye was administered preoperatively both through oral and subcutaneous routes, and the peritoneal cavity was explored using laparoscopy. This laparoscopic lymphoid dye test precisely identified the area of chylous leakage, and the authors were able to repair the malformed lymphatic duct directly at laparotomy. Both postoperative courses have been favorable with no recurrence of symptoms. The lymphatic duct ligation should be considered in cases resistant to conservative treatment for over a month. The present laparoscopic lymphoid dye test is a novel and useful procedure that allows surgeons to identify the exact location of chylous leakage, and thus successfully ligate the lymphatic duct.

Physicochemical properties and structure of large, medium and small granule starches in fractions of normal barley endosperm.

Normal barley grain was milled to flour with a machine used to polish brewers' rice from the surface layer to the center. Large (18.4 microm, median size), medium (12.3 microm) and small (2.2 microm) granule starches were isolated from classified flours. Their physicochemical properties and fine structure were investigated. The percentage (w%) of large granules decreased from the surface layer to the center, while the amounts of medium and small granules increased. Although all the starch granules were an A-type crystal, the relative crystallinity varied from 22.0 to 27.4%. The DPn of the amyloses was around 1600 and similar for all the samples. But the amylose content of the starches varied from 21.9 to 26.4%. Also, the amylopectins showed differences in DPn (around 5700-7900) and chain-length distribution between granule size or fractions. The transition temperature ranges and the enthalpy values of the starch granules differed with granule size. The gelatinization properties showed no correlation with any of the parameters, except the enthalpy value and relative crystallinity (gamma = +0.73). The findings suggested that the structural characteristics of the starches in classified flours of normal barley differed essentially from those of waxy barley.

Intralobar bronchopulmonary sequestration evaluated by contrast-enhanced three-dimensional MR angiography.

Bronchopulmonary sequestration (PS) is characterized by non-functioning lung tissue fed from one or several aberrant systemic arteries. The condition is diagnosed by visualizing the feeding arteries using non-invasive CT, MRI, colour Doppler sonography or conventional angiography. We present a 5-year-old boy in whom intralobar sequestration was diagnosed using contrast-enhanced 3D MR angiography, which visualised fine blood vessels in the thoraco-abdominal region without arterial puncture. This technique is useful for diagnosing PS.

Characterization of thiamin-binding protein from buckwheat seeds.

A thiamin-binding protein from buckwheat (Fagopyrum esculentum Moench) seeds gave two bands of 56-and 50-kDa in the absence of 2-mercaptoethanol and a single band of 25-kDa in the presence of 2-mercaptoethanol on sodium dodecylsulfate gel electrophoresis. These results indicate that the protein consists of polypeptides linked by disulfide bond(s). The protein isolated from buckwheat seeds did not have immunological homology with the thiamin-binding proteins from rice seeds and sesame seeds. However, the binding of the protein to thiamin was inhibited by the modification of the carboxyl residues in the protein as well as that of the thiamin-binding protein from rice seeds. These results suggest that the thiamin-binding protein from buckwheat seeds differ from those from rice seeds and sesame seeds as to subunit structure or immunological properties, but resembles them in the mechanism of binding thiamin.

Thiamin-binding protein from sunflower seeds.

A thiamin-binding protein was isolated from sunflower seeds. Its molecular mass was estimated to be 230 kDa by gel filtration. The protein was suggested to be composed of six subunits, which consisted of polypeptides linked by disulfide bond(s). The protein contained a large amount of glutamine or glutamic acid (19.9 mol%) and asparagine or aspartic acid (11.1 mol%). The levels of tryptophan and valine in the protein were low. These properties of the thiamin-binding protein were similar to those of helianthinin. Optimum pH for the thiamin-binding activity of the protein was 8.0 to 9.0. The thiamin-binding activity was not inhibited by thiamin monophosphate, thiamin pyrophosphate, oxythiamin, or pyrithiamin. These properties of the thiamin-binding protein from sunflower seeds were similar to those from buckwheat seeds, but not to those from rice seeds and sesame seeds.

Characterization, gene cloning and expression of isocitrate lyase involved in the assimilation of one-carbon compounds in Hyphomicrobium methylovorum GM2.

Isocitrate lyase of an obligate methylotrophic bacterium, Hyphomicrobium methylovorum GM2, was purified to homogeneity and characterized. The enzyme is a homotetramer of identical 62-kDa subunits. After the enzyme had been incubated at temperatures up to 25 degrees C for 30 min, no loss of activity was observed. The enzyme was stable in the pH range of 7.5-9.0. Maximum activity was observed at pH 7.5 and around 45 degrees C. The Km value for Ds-isocitrate was 0.51 mM. The activity required Mg2+ and was inhibited by oxalate, succinate and glycolate. The gene encoding the isocitrate lyase and its flanking regions were isolated from H. methylovorum GM2. Nucleotide sequencing of recombinant plasmids revealed that the isocitrate lyase gene codes for a 540-amino-acid protein. The amino acid sequence of the enzyme is similar to those of the enzymes from Escherichia coli (40% identity) and cucumber (37% identity). The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli HB101. The transformed E. coli cells expressed isocitrate lyase, which was indistinguishable from the purified H. methylovorum GM2 isocitrate lyase on analysis by SDS/PAGE.

Cloning and analysis of methanol oxidation genes in the methylotroph Hyphomicrobium methylovorum GM2.

The gene encoding the alpha-subunit of methanol dehydrogenase (mxaF) and its flanking region was isolated from a methylotrophic bacterium, Hyphomicrobium methylovorum GM2. The deduced amino acid sequence of MxaF showed 80, 80, 74 and 66% identity with those of Methylobacterium extorquens AM1, M. organophilum XX, Paracoccus denitrificans and Methylophilus methylotrophus, respectively. The putative mxaF promoter sequence (-35 -AAAGACA-, -10 -TAGAA-) observed in other methylotrophs was not found in the region 5' of H. methylovorum GM2 mxaF. Downstream of mxaF, five open reading frames and one partial open reading frame were detected which had high identity with the genes mxaJ, mxaG, mxaI, mxaR, mxaS and mxaA. This indicated the existence of a mxaFJGIRSA gene cluster in H. methylovorum GM2, as previously observed in M. extorquens AM1.

Cloning and expression of the gene for serine-glyoxylate aminotransferase from an obligate methylotroph Hyphomicrobium methylovorum GM2.

The gene encoding serine-glyoxylate aminotransferase, one of key enzymes for the assimilation of one-carbon compounds in methylotrophs, and its flanking regions were isolated from an obligate methylotrophic bacterium, Hyphomicrobium methylovorum GM2. Nucleotide sequencing of the recombinant plasmids revealed that the serine-glyoxylate aminotransferase gene encodes a 405-amino-acid protein with a calculated molecular mass of 43880 Da. The amino acid sequence of the enzyme showed identity to the sequences of serine-glyoxylate aminotransferase of Methylobacterium extorquens AM1 (57%), aspartate aminotransferase of Methanobacterium thermoformicicum (31%), human peroxisomal alanine-glyoxylate aminotransferase (27%), and serine-pyruvate aminotransferase of rat liver mitochondria (33%). The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector pKK223-3, was introduced into Escherichia coli HB101. The recombinant enzyme was purified from transformed E. coli cells and analyzed by immunological and enzymological methods. The overexpressed enzyme was indistinguishable from the wild-type enzyme isolated from H. methylovorum GM2.

Efficient L-serine production from methanol and glycine by resting cells of Methylobacterium sp. strain MN43.

Resting cells of methanol-utilizing microorganisms isolated from soils were examined for L-serine production under conditions in which L-serine-degradation was suppressed. Strain MN43, a facultative methylotrophic bacterium identified as a Methylobacterium sp., was selected for further studies. Under the optimal conditions, 65 mg/ml L-serine was produced by this bacterium from 50 mg/ml glycine and 104 mg/ml methanol in 5 days, with a molar conversion ratio from glycine to L-serine of 93%. This production is the highest so far reported for microbes producing L-serine.

Peroxidase-catalyzed generation of catechin oligomers that inhibit glucosyltransferase from Streptococcus sobrinus.

Oolong tea extract (OTE) and the purified polymeric polyphenols from OTE have been found to inhibit glucosyltransferase (GTase) of mutans streptococci. In view of the partial fermentation characteristic of oolong tea, we describe here an in vitro model reaction system to produce partially fermented products of D-(+)-catechin or green tea extract (GTE) using horseradish peroxidase. A dimeric catechin molecule was identified as dehydro-dicatechin A by instrumental analyses. The molecular size of some oligomeric catechins was estimated by the elution profile with HPLC. These catechin oligomers markedly inhibited GTase from Streptococcus sobrinus 6715. As the degree of polymerization of catechin or GTE increased, GTase was inhibited more effectively. These results suggest that polymeric polyphenols found in OTE are synthesized by partial fermentation due to oxidases/peroxidases present in tea leaves.

Immunological characterization of serine-glyoxylate aminotransferase and hydroxypyruvate reductase from a methylotrophic bacterium, Hyphomicrobium methylovorum GM2.

Immunological characterization of serine-glyoxylate aminotransferase and hydroxypyruvate reductase, key enzymes for the assimilation of one-carbon compounds in methylotrophs, was performed using antibodies raised against these enzymes purified from Hyphomicrobium methylovorum GM2. Immunodiffusion studies indicated that serine-glyoxylate aminotransferase and hydroxypyruvate reductase of all seven Hyphomicrobium strains tested were immunochemically similar. In immunotitration experiments and Western blot analyses of both enzymes in the genera Hyphomicrobium and Methylobacterium, the serine-glyoxylate aminotransferase of the genus Methylobacterium exhibited low similarity to that of the genus Hyphomicrobium. For hydroxypyruvate reductase, no immunological relationship was observed between the genera Hyphomicrobium and Methylobacterium, which was in agreement with the differences in primary structure and enzymological properties.

Isolation of a Thiamine-binding Protein from Rice Germ and Distribution of Similar Proteins.

A thiamine-binding protein was purified from rice germ (Oryza sativa L.) by extraction, salting-out with ammonium sulfate, and column chromatography. From the results of molecular mass, Kd and Bmax values for thiamine-binding, binding specificity for thiamine phosphates and analog, the protein was suggested to be identical to the thiamine-binding protein in rice bran. The thiamine-binding protein w as more efficiently purified from rice germ than from rice bran. The protein was rich in glutamic acid (and/or glutamine) and glycine. The protein did not show immunological similarity to thiamine-binding proteins in buckwheat and sesame seeds. However proteins similar to the thiamine-binding protein from rice germ existed in gramineous seeds. They were suggested to have thiamine-binding activity and to be of the same molecular mass as the thiamine-binding protein.

Cloning and expression of the gene for hydroxypyruvate reductase (D-glycerate dehydrogenase from an obligate methylotroph Hyphomicrobium methylovorum GM2.

The gene encoding hydroxypyruvate reductase, catalyzing the asymmetric reduction of hydroxypyruvate to D-glycerate, and its flanking regions were isolated from a methylotrophic bacterium, Hyphomicrobium methylovorum GM2. Nucleotide sequencing of the recombinant plasmids revealed that the hydroxypyruvate-reductase gene codes for the 322-amino-acid protein with calculated molecular mass 35,726 Da. The sequence was confirmed by sequencing the intact enzyme and peptides obtained by digestion of the enzyme with Achromobacter proteinase I. The amino acid sequence of the enzyme showed similarity to members of the D-isomer-specific 2-hydroxyacid dehydrogenase family. The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector pKK223-3, was introduced into Escherichia coli HB101. The recombinant enzyme purified from the transformed E. coli cells was indistinguishable from the enzyme isolated from H. methylovorum GM2 by immunological and enzymological analyses.

The vascular tissue angiotensin I-converting enzyme activity and aortic elastin content in stroke-prone spontaneously hypertensive rats fed fish diet.

1. Stroke-prone spontaneously hypertensive rats (SHRSP) were fed a diet with fish meal as the protein source (fish diet) during the progressive stage of hypertension, and its effects on the activity of angiotensin I-converting enzyme (ACE) in serum and vascular tissues and on the aortic elastin content were studied. The effects of the antihypertensive drugs captopril and hydralazine were also studied. 2. Stroke-prone spontaneously hypertensive rats fed the fish diet showed a distinctly lower level (P < 0.05) of serum ACE activity than the control group fed a commercial stock chow. 3. ACE activity was enhanced in the SHRSP which was administered with captopril. 4. Serum ACE activity was similar in the SHRSP receiving the hydralazine treatment and the control group. 5. The thoracic aorta ACE activity was lowered more (P < 0.05) in the fish diet group and the captopril-treated group than in the control group. In the hydralazine-treated group however, the activity was similar to the control group. 6. The ratio of aorta weight to bodyweight was significantly lower (P < 0.05) in the fish diet group and the captopril-treated group than in the control group, but there was no difference in the hydralazine group. Higher levels of aortic elastin were observed in the drug-treated groups (P < 0.05). 7. No differences were seen between the fish diet and captopril-treated groups by electron-microscopy. 8. The results suggest that suppression of hypertrophy and ameliorations of reduction in elasticity of the vascular wall in the SHRSP fed a fish diet were due to inhibition of vascular tissue ACE activity.

L-serine production by a methylotroph and its related enzymes.

The production process of L-serine from methanol and glycine has been developed using a methylotroph with the serine pathway. Consecutive reactions of two enzymes, methanol dehydrogenase (MDH) and serine hydroxymethyltransferase (SHMT) are involved in the production. We screened a high producer, Hyphomicrobium methylovorum, which is an obligate methylotroph. With resting cells of the bacterium, 24 mg/ml of L-serine was produced from 100 mg/ml of glycine and 48 mg/ml of methanol in 3 days under optimal conditions. Next, a glycine-resistant mutant GM2 showed improved serine production (32-34 mg/ml). The mutant GM2 was found to have elevated activities of MDH and SHMT. Since there has so far been little report on the systematic characterization of enzymes of the serine pathway in methylotrophs, not only the above two enzymes but also the other three enzymes in H. methylovorum were purified and characterized: MDH, SHMT and hydroxypyruvate reductase (HPR) were crystallized; serine-glyoxylate aminotransferase (SGAT) and glycerate kinase (GK) were purified to homogeneity. As a result, all these enzymes were found to be stable against preservation and to exist abundantly in the bacterium. The gene of SHMT was cloned and its deduced amino acid sequence had homology to those of Escherichia coli (55%) and rabbit liver (44%), whereas the enzyme of the bacterium was immunochemically distinguishable from those of microorganisms other than Hyphomicrobium strains and mammalian livers.

Molecular cloning and expression of the gene for serine hydroxymethyltransferase from an obligate methylotroph Hyphomicrobium methylovorum GM2.

The gene encoding serine hydroxymethyltransferase (SHMT), one of the key enzymes of the one-carbon-compound assimilation of a methylotroph, Hyphomicrobium methylovorum GM2, and its flanking regions were isolated using a DNA fragment encoding Escherichia coli SHMT as a probe. Nucleotide sequencing of the recombinant plasmids revealed the SHMT gene codes for the 434-amino-acid protein with a calculated molecular mass of 46,068 Da. The amino-acid sequence of the enzyme showed identity to the sequences of the enzymes from E. coli (55%) and rabbit liver (44%). The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector pKK223-3, was introduced to an SHMT-deficient E. coli mutant ME5427 (glyA-). The transformed E. coli cells expressed SHMT, which was immunologically and enzymologically indistinguishable from the enzyme isolated from H. methylovorum GM2.

Enzymatic assay for L-serine and glyoxylate involving the enzymes in the serine pathway of a methylotroph.

An easy, rapid, and accurate enzymatic assay method for L-serine was established involving two enzymes, serine-glyoxylate aminotransferase (SGAT, EC 2.6.1.45) and hydroxypyruvate reductase (HPR, EC 1.1.1.81), in the serine pathway of the methylotrophic bacterium, Hyphomicrobium methylovorum (IFO 14180), from which they were purified. This method consists of two reaction steps: the first is the nearly irreversible transamination of L-serine and glyoxylate by SGAT, and the second is the HPR reaction involving NADH, which comprises the absolutely irreversible reduction of hydroxypyruvate derived from L-serine by SGAT. The amounts of L-serine were determined spectrophotometrically as the decrease in the amount of NADH. When the values determined with the present enzymatic method were compared with those obtained with an amino acid analyzer, the correlation coefficient was found to be 0.9963. This method can also be applied to the assaying of glyoxylate.