Folate intakes and folate biomarker profiles of pregnant Japanese women in the first trimester.

OBJECTIVE: To assess the status of dietary folate intake, serum and red blood cell (RBC) folate, and related nutritional biomarkers in healthy Japanese women in early pregnancy. DESIGN: A cross-sectional, observational study. SUBJECTS: Pregnant women in the first trimester, at 7-15 weeks gestation (n=70), who were not consuming any folate supplements or folate fortified foods. METHODS: Three-day dietary records were obtained from each subject to assess dietary folate intake. Blood samples were collected for measurement of biomarkers. Biomarkers and nutrient intake were analyzed in two groups defined by their serum folate concentrations: the low folate group (serum folate < 9 ng/ml) and the high folate group (serum folate > or = 9 ng/ml). RESULT: Mean serum and RBC folate concentrations in all subjects were 10.3 and 519 ng/ml, respectively. These levels were remarkably higher than the reported values from many other countries despite our subjects receiving no folic acids supplements. However, mean folate intake by our subjects from natural foods was 289 microg/day, which is thought to be low according to the Japanese dietary recommendation specified for pregnant women. The intake of spinach and fruits was significantly greater in the high folate group than in the low folate group. CONCLUSION: Folate intake was thought to be adequate to maintain a desirable level of serum folate concentration in Japanese pregnant women in the first trimester, although the intake of folate from natural food was not high enough to meet the recommended daily intake.

Enhanced tumor cell selectivity of adriamycin-monoclonal antibody conjugate via a poly(ethylene glycol)-based cleavable linker.

A novel linker consisting of poly(ethylene glycol) (PEG) and dipeptide was used for conjugation of adriamycin with tumor-specific monoclonal antibody, NL-1, to confirm that the linker can be cleaved selectively with the tumor specific enzyme to express cytotoxicity of the anti-tumor agent. Initially, adriamycin-conjugated PEG linkers through different amino acid compositions, alanyl-valine (Ala-Val), alanyl-proline (Ala-Pro), and glycyl-proline (Gly-Pro) sequences, were prepared to confirm selective digestion with model enzymes. Adriamycin was released by particular model endoproteases, thermolysin and proline endopeptidase, from the linkers with different efficiency. When conjugates were prepared using these adriamycin-bound linkers, conjugates had no loss of binding affinity and specificity for common acute lymphoblastic leukemia antigen (CALLA) expressed on the Daudi cell surfaces as the target of NL-1 antibody. In addition, adriamycin release from the conjugates was also confirmed by incubating them with specific proteases. Tumor cell growth was inhibited dose-dependently for the conjugates carrying Ala-Val and Gly-Pro linkers, whereas significant inhibitory effect was abolished for the conjugate carrying Ala-Pro linker, indicating that cytotoxic effect can be controlled by specificity of antibody and composition of linker peptide. IC(50) for Ala-Val linked conjugate was approximately 3.5 microg/ml and that for Gly-Pro linked conjugate was 5.2 microg/ml. PEG-dipeptidyl linker demonstrated here will be an effective tool for the preparation of immunoconjugate, especially specific activation of anti-tumor agents at desired tumor tissues.

Sesquiterpene coumarins from the roots of Ferula assa-foetida.

From the chloroform extract of the roots of Ferula assa-foetida, two sesquiterpene coumarins designated assafoetidnol A and assafoetidnol B were isolated, in addition to six known compounds, gummosin, polyanthin, badrakemin, neveskone, samarcandin and galbanic acid. The structures of the sesquiterpenes new compounds were established by spectroscopic methods.

Monoterpene coumarins from Ferula ferulago.

Two monoterpene coumarins, designated ferulagol A and B, as well as three known monoterpene coumarins and three sesquiterpene lactones were isolated from the chloroform extract of the roots of Ferula ferulago. The structures of ferulagol A and B were determined to be 7-[(E)-3'-hydroxy-3',7'-dimethyl-4',6'-octadienyloxy]coumarin and 7-[(3'Z,5'E)-7'-hydroxy-3',7'-dimethyl-3',5'-octadienyloxy]coumarin, respectively.

An eudesmanolide and a carotane from Ferula sinaica.

Re-examination of the chemical constituents of the leaves of Ferula sinaica afforded a new eudesmanolide and a new carotane. The structures were elucidated by spectroscopic methods.

The first total synthesis of (+/-)-linderol A, a tricyclic hexahydrodibenzofuran constituent of Lindera umbellata bark, with potent inhibitory activity on melanin biosynthesis of cultured B-16 melanoma cells.

[reaction in text] The first total synthesis of (+/-)-linderol A, a hexahydrodibenzofuran isolated from Lindera umbellata bark, with potent inhibitory activity on melanin biosynthesis of cultured B-16 melanoma cells was achieved via a 20-step of reaction in 7.64% overall yield starting from 4,6-dimethoxysalicylaldehyde.

Genome relationships in the genus Dasypyrum (Gramineae).

To elucidate the genome relationships in the genus Dasypyrum and the ancestry of tetraploid D. breviaristatum, two cytotypes of D. breviaristatum and D. villosum were reciprocally crossed with one another. Chromosome pairing at the first metaphase of meiosis and fertility were examined in the F1 hybrids and the parental plants. The mean pairing configuration and mean arm pairing frequency in D. villosum-D. breviaristatum (2x) hybrids were 11.12I + 1.44II per cell and 0.107, respectively, and they were almost completely sterile. In D. breviaristatum (4x)-D. breviaristatum (2x) hybrid, up to seven trivalents were formed, and the mean pairing configuration was 3.38I + 3.20II + 3.74III + 0.005IV per cell. The mean arm pairing frequency and relative affinity calculated in that F1 hybrid were 0.915 and 0.641, respectively. Seven bivalents and seven univalents were characteristically formed in D. villosum-D. breviaristatum (4x) hybrids. Based on the present results, we clearly concluded that the genome of diploid D. breviaristatum is distantly related to the genome V of D. villosum, and that these two species have different basic genomes. We, therefore, proposed the symbol Vb for the haploid genome of diploid cytotype of D. breviaristatum. Moreover, we concluded that tetraploid D. breviaristatum is an autotetraploid with doubled sets of the genomes homologous with that of diploid D. breviaristatum, and we proposed the genome constitution VbVb for the haploid genome set of tetraploid cytotype of D. breviaristatum. Furthermore, from the chromosome pairing in the F1 hybrids involving Moroccan and Greek accessions, it was suggested that complicated rearrangements of chromosome structure have occurred in tetraploid D. breviaristatum in its natural populations across the entire distribution area.

The optimal stimulation site for chronic stimulation of the subthalamic nucleus in Parkinson's disease.

To determine the optimal stimulation site within the subthalamic nucleus (STN), monopolar stimulation of four electrode contacts and the resulting effects on parkinsonian symptoms were evaluated in 10 consecutive patients. The UPDRS score for rigidity and akinesia improved significantly after stimulation at each of the contacts, compared to the pre-evaluation state (Fisher's test, p < 0.05). The most significant improvement was obtained after stimulation at contact-2 (rigidity: 74.4 +/- 20.4%, akinesia: 53.7 +/- 14.3%) (Fisher's test, p < 0.001). Contact-2 was located at the dorsal border of the STN at a mean distance of 0.3 +/- 0.7 mm. DBS at the dorsal border of the STN, where the stimulation affects the neurons as well as their axonal fibers, produces the greatest clinical improvement in parkinsonian symptoms.

Variation and geographical distribution of the genotypes controlling the diagnostic spike morphology of two varieties of Aegilops caudata l.

Aegilops caudata L. is an annual wild relative of wheat distributed over the northeastern Mediterranean basin. It consists of two taxonomic varieties, var. typica with awnless lateral spikelets and var.polyathera with awned lateral spikelets. To clarify the variation and the geographical distribution of the genotypes controlling the diagnostic spike morphology of the two taxonomic varieties, three crossing experiments were carried out. First, two varieties collected from nine sympatric populations in the Aegean islands were crossed reciprocally. All of the F1 hybrids were var. typica and the segregation ratio in the F2 generation was 3 typica: 1 polyathera. Secondly, 13 typica accessions collected from the entire distribution area of the variety were crossed with a common polyathera accession. The F1 hybrids involving eight typica accessions from Greece and West Anatolia were var. typica, while those involving five typica accessions from East Anatolia, Syria and Iraq were var. polyathera. Thirdly, the typica F1 hybrids between the Aegean and the Syrian typica accessions were backcrossed to the latter. Four of the seven BC1F1 plants obtained were var. typica, but the other three were var. polyathera. Based on these results, the following two conclusions were made. First, the awnless lateral spikelets characteristic of var. typica are due to two different genotypes: one is a dominant allele suppressing awn development on lateral spikelets and the other is a recessive allele(s) for awnless lateral spikelets with no dominant suppressor allele. Secondly, the former genotype occurs only in the western region of the distribution area of the species, while the latter occurs in the eastern region. The present results and the recent palaeopalynological evidence also suggested that var. polyathera, with more awns than var. typica, rapidly colonized Central Anatolia from the Levant or East Taurus/Zagros mountains arc after the last glacial period.

Genetic differentiation and post-glacial establishment of the geographical distribution in Aegilops caudata L.

Aegilops caudata L. is a diploid wild relative of wheat distributed over the north-eastern Mediterranean from Greece to northern Iraq. To elucidate the geographical differentiation pattern, 35 accessions derived from the entire distribution area were crossed with four Tester strains. Pollen fertility in the F1 hybrids varied from 0 to 96.3% among cross combinations, closely correlating with the geographical regions where the parental accessions were collected. Based on the intraspecific hybrid sterility, the present distribution area of Ae. caudata was divided into two geographical regions effectively isolated by the mountainous region lying between West Anatolia and Central Anatolia. The western region is composed of Greece and West Anatolia, while the eastern region consists of Central Anatolia, South Anatolia, East Anatolia and northern Iraq. The present results and the facts from recent palaeopalynological works suggest that during the maximum glacial period from 18,000 BP to 16,000 BP, Ae. caudata occurred in the two isolated regions, i.e., the region surrounding the Aegean Sea and the western Levant or some sheltered habitats in the East Taurus/Zagros mountains arc, and that it migrated into Central and East Anatolia from the latter regions as the climate became warmer. Furthermore, it is also suggested that the Levant populations now occur in the eastern region of the distribution, while those occurring in the Aegean Sea region during the last glacial period now occupy the western region of the distribution.

Reduction of hydroxamic acids to the corresponding amides catalyzed by rabbit blood.

1. The hydroxamic acids N-hydroxyphenacetin and N-hydroxy-2-acetylaminofluorene were reduced to the corresponding amides, phenacetin and 2-acetylaminofluorene respectively by rabbit blood supplemented with both NAD(P)H and FAD. These reducing activities were found in erythrocytes but not in plasma, and were sensitive to inhibition by carbon monoxide and oxygen. When blood or erythrocytes were boiled, these activities were not abolished. 2. Haemoproteins such as haemoglobin and catalase exhibited the reductase activity in the presence of both NAD(P)H and FAD under anaerobic conditions. The activity was not abolished when the haemoproteins were boiled. 3. Haematin showed a significant reducing activity in the presence of these cofactors. The activity of haematin was also observed with the photochemically reduced form of FAD. 4. The reduction system in blood was composed of NAD(P)H, FAD and haemoglobin. Reduction appears to proceed in two steps, i.e. the reduction of FAD by NADH or NADPH, followed by the non-enzymatic reduction of the hydroxamic acids to the amides by reduced FAD, catalyzed by the haem group of haemoglobin in rabbit erythrocytes.

In vivo antitumor effects of electrochemotherapy in a tongue cancer model.

PURPOSE: This study investigated the in vivo antitumor effects of electrochemotherapy (ECT) using electroporation and bleomycin in a hamster tongue cancer model to assess its clinical applicability. MATERIALS AND METHODS: Twenty animals with chemically induced tongue cancer were divided into four experimental groups designated B-E-, B-E+, B+E-, and B+E+. The B+E+ and B+E- groups received an intraperitoneal injection of 100 microg bleomycin. Fifteen minutes after the injection, the B+E+ animals received electric pulses. The B-E+ group received only electric pulses. The B-E- group received neither bleomycin nor electric pulses. Each group received the same treatment twice. The antitumor effects were assessed based on tumor volume reduction and histologic findings. RESULTS: The B+E+ group showed remarkable tumor volume reduction, decreasing an average to 8.8% of its original volume 14 days after the treatment. Complete loss of the protruding tumor was observed in two of the five animals. Histologically, the tumors of the B+E+ group consisted of severely degenerated tumor cells and desquamative keratinizing cells. No living cancer cells were detected in three animals. The B+E-, B-E+, and B-E- groups showed progressive tumor growth, exceeding 200% of initial tumor volume during the experimental period. CONCLUSION: The current study showed remarkable antitumor effects of ECT with bleomycin in the hamster tongue cancer model. ECT with bleomycin may be clinically applicable to the treatment of oral cancer.

Subthalamic nucleus stimulation for gait disturbance in Parkinson's disease.

OBJECTIVE: A preliminary study of subthalamic nucleus (STN) stimulation was performed to determine its applicability for the treatment of gait and postural disturbances in Parkinson's disease. METHODS: Five Parkinson's disease patients with freezing gait and postural instability were selected for this study. Their ages ranged from 60 to 73 years (mean+/-standard deviation, 65.6+/-4.8 years). Semi-microelectrode recording was used to identify the STN and to place a chronic electrical stimulation electrode within the right STN in all patients. The Unified Parkinson's Disease Rating Scale and the modified Hoehn and Yahr Staging Scale were used to assess patients in on- and off-drug conditions before surgery and 3 months after surgery. RESULTS: The scores on the Hoehn and Yahr Staging Scale and the total Unified Parkinson's Disease Rating Scale for akinesia (P < 0.05), gait (P < 0.05), and gait and posture (P < 0.01) in off-drug on-stimulation conditions significantly improved over the preoperative and postoperative off-drug off-stimulation conditions (analysis of variance [ANOVA], P < 0.01). Improvement over the preoperative scores was 24% on the Hoehn and Yahr Staging Scale, 43.6% on the total Unified Parkinson's Disease Rating Scale, 33.4% for akinesia, 36.6% for gait, and 38.7% for gait and posture. However, stimulation in the on-drug phase did not show a significant difference compared with pre- and postoperative conditions (ANOVA, P > 0.05). Comparisons between preoperative on-drug and postoperative off-drug on-stimulation conditions revealed that there were no significant differences in the scores, except for gait (ANOVA, P < 0.05). The scores on subscales for falling, freezing, walking, and gait in off-drug on-stimulation conditions were significantly improved over the scores for preoperative and postoperative off-stimulation (ANOVA, P < 0.05), but the score for postural stability remained unchanged. CONCLUSION: Our findings showed that STN stimulation effectively alleviates freezing gait and improves walking to its status during the preoperative on-drug phase and can be applied for treatment of Parkinson's disease patients with these symptoms.

Debromination of (alpha-bromoiso-valeryl)urea catalysed by rat blood.

(Alpha-bromoiso-valeryl) urea, a sedative or hypnotic, is metabolized to (3-methylbutyryl)urea by reductive debromination. This study was designed to evaluate the role of blood in the debromination of (alpha-bromoiso-valeryl) urea. Rat blood containing an electron donor had significant debrominating activity toward (alpha-bromoiso-valeryl)urea. This debromination proceeded by enzymatic and non-enzymatic processes which required both NADH (or NADPH) and flavin mononucleotide (FMN), under anaerobic conditions. The debrominating activity was sensitive to inhibition by carbon monoxide, and the pH optimum was 8.5. When FMN was replaced by flavin adenine dinucleotide (FAD) or riboflavin, similar results were obtained. The optimum concentration of flavins was 10(-4) M. The reductive debromination was also mediated by rat erythrocytes, but not by plasma. When the blood or erythrocytes were boiled, the debrominating activity was not abolished, but was enhanced, suggesting that the activity arises from the haemoglobin in erythrocytes, and haemoglobin had debrominating activity when supplemented with both a reduced pyridine nucleotide and a flavin. Furthermore, haematin had significant debrominating activity in the presence of these cofactors. The activity of haematin was also observed with the photochemically reduced form of FMN. The results imply that the debromination proceeds in two steps--enzymatic or non-enzymatic reduction of a flavin such as FAD, FMN or riboflavin by NADPH or NADH, then non-enzymatic reductive debromination of (alpha-bromoiso-valeryl)urea to (3-methylbutyryl)urea catalysed by the haem group of rat haemoglobin in the presence of the reduced flavin.

Hyaluronan activates mitogen-activated protein kinase via Ras-signaling pathway.

Hyaluronan (HA) triggers a wide variety of cellular functions, yet its signaling pathway remains largely unclear. We found that HA-treatment of 3Y1 cells activated tyrosine phosphorylation of cellular proteins and mitogen-activated protein (MAP) kinase in a time- and dose-dependent manner, and, subsequently, stimulated cell growth. This HA-activity was resistant to boiling at 100 degrees C but completely abolished by treatment with hyaluronidase, suggesting that HA itself, but not any HA-associated proteins, has the activity. In addition, we found that HA-dependent activation of MAP kinase was strongly suppressed by the expression of dominant negative ras (S17N ras). These results suggest that Ras-MAP kinase pathway is activated by HA and may play an important role in HA-dependent signaling.

Changes in protein synthesis and calcium homeostasis in the thalamus of spontaneously hypertensive rats with focal cerebral ischemia.

The thalamus has been shown to undergo secondary degeneration after cerebrocortical ischemia. However, little is known about the time course of the retrograde thalamic degeneration. The present study was designed to investigate time-dependent changes in the morphology, protein synthesis and calcium metabolism of thalamic neurons in middle cerebral artery (MCA)-occluded spontaneously hypertensive stroke-prone rats that showed primary focal ischemia in the temporoparietal cortex after permanent occlusion of the left distal MCA. In the histologic study by light and electron microscopy, swelling of the nucleus and shrinkage of the perikarya were seen in some neurons of the ventroposterior (VP) thalamic nucleus on the lesioned side at 5 days after ischemia. At the same time, the incorporation of radiolabeled leucine in VP thalamic neurons began to decrease significantly with concomitant a decrease in the number of polyribosomes in the neurons. Conspicuous 45Ca accumulation was noted at 3 days after ischemia and persisted up to 1 month in the VP thalamic nucleus on the lesioned side. These findings suggest that the secondary thalamic degeneration after cortical infarction starts with disruption of calcium homeostasis in situ at the third day after MCA occlusion, followed by a decrease in polyribosomes but not by disaggregation of polyribosomes as seen in hippocampal CA1 neurons subjected to transient forebrain ischemia.

Neural activity of the subthalamic nucleus in Parkinson's disease patients.

The neural activity pattern of the subthalmic nucleus (STN) was investigated in five patients with Parkinson's disease who were scheduled for electrode implantation for chronic stimulation of the STN. The initial target was placed 8 mm or 10 mm lateral to the midline, 3 mm to 4 mm posterior to the midcommissural point, and 5 mm to 6 mm below the intercommissural (AC-PC) line. The STN was identified by semi-microelectrode recordings with a trajectory moving laterally in 2-mm steps. The amplitudes of multi-unit activities were relatively low at depths from 8 mm to 5 mm above and from 1 mm to 4 mm below the target, while those 4 mm to 0 mm above the target were significantly higher than at the other sites (ANOVA, Fisher's test, p < 0.05), with the highest amplitude at 2 mm above the target (91.0 +/- 23.3 mu v, n = 15). In the mediolateral direction, amplitudes were relatively higher in the lateral portion, and amplitudes at 14 mm lateral to the midline were significantly higher than at the other sites (ANOVA, Fisher's test, p < 0.05). The target for chronic electrical stimulation was determined to be at the midpoint of the hyperactive STN, i.e., 12 mm lateral to the midline in three patients and 13 mm lateral in two patients. Movement-related neural activity was observed at 5 sites, i.e., 3 sites responded to passive movement of the contralateral wrist and 2 sites to passive knee and/or ankle movement. In conclusion, our data show that the lateral part of the STN is hyperactive in PD, and recordings of neural activities contributed greatly to identifying the STN and determining the target for chronic stimulation within it.

Quinone-dependent tertiary amine N-oxide reduction in rat blood.

Rat blood exhibited a significant quinone-dependent N-oxide reductase activity towards imipramine N-oxide. The reduction mediated by the blood proceeded in the presence of both NAD(P)H and menadione under anaerobic conditions. When menadione was replaced with 1,4-naphthoquinone or 9,10-phenanthrenequinone, similar results were obtained. The reduction was also mediated by the combination of rat erythrocytes and plasma. The reducing activity was inhibited by dicumarol and carbon monoxide. When boiled plasma was combined with untreated erythrocytes, the N-oxide reducing activity was abolished. In contrast, when boiled erythrocytes were combined with untreated plasma, the activity was unchanged. These results suggest that the activity is caused by the heme of hemoglobin in erythrocytes and quinone reductase in plasma. In fact, erythrocytes and hemoglobin have the ability to reduce the N-oxide when supplemented with DT-diaphorase purified from rat liver in the presence of both NAD(P)H and menadione. Hemoglobin also exhibits N-oxide reductase activity with reduced menadione (menadiol). Furthermore, hematin exhibits a significant reducing activity in the presence of menadiol. The reduction appears to proceed in two steps. The first step is enzymatic reduction of quinones to dihydroquinones by quinone reductase(s) with NADPH or NADH in plasma. The second step is nonenzymatic reduction of imipramine N-oxide to imipramine by the dihydroquinones, catalyzed by the heme group of hemoglobin in erythrocytes. Cyclobenzaprine N-oxide and brucine N-oxide are similarly transformed to the corresponding amines by the above reducing system in blood. These results suggest that blood plays an important role in the reduction of tertiary amine N-oxides to tertiary amines.

Protein synthesis inhibitor transiently reduces neuronal death in the thalamus of spontaneously hypertensive rats following cortical infarction.

Using stroke-prone spontaneously hypertensive rats with permanent occlusion of the middle cerebral artery (MCA), we investigated whether the secondary thalamic degeneration following cortical infarction is related to apoptosis, and whether the protein synthesis inhibitor cycloheximide (CHX) ameliorates this degenerative process. TdT-mediated dUTP-biotin nick end labeling (TUNEL staining) revealed a distinct pattern of nuclear staining in many ventroposterior (VP) thalamic nucleus neurons on the lesioned side at 1 week after MCA occlusion. In rats with a single or continuous intraventricular infusion of CHX, starting just after brain ischemia, in the VP thalamic neurons were significantly more numerous than those in the thalamic nucleus of rats with vehicle infusion at 1 week after MCA occlusion. However, at 2 weeks after MCA occlusion, the numbers of VP thalamic neurons were similar in the CHX- and vehicle-treated groups. These findings suggest that the secondary thalamic degeneration following cortical infarction is an event reminiscent of apoptosis and that CHX prevents the secondary thalamic neuronal death transiently.

Increased expression of carnitine palmitoyltransferase I gene is repressed by administering L-carnitine in the hearts of carnitine-deficient juvenile visceral steatosis mice.

The juvenile visceral steatosis (JVS) mouse is a novel mutant animal for studying systemic carnitine deficiency. The importance of the model has been pointed out in carnitine-deficient cardiac hypertrophy, since cardiomyopathy has been often improved after oral carnitine therapy in human systemic carnitine deficiency. To understand the effects of carnitine deficiency on gene expression in the heart, we tried to find the genes regulated by carnitine by means of a modified differential display procedure. Carnitine palmitoyltransferase I (CPT I) was one of the isolated genes. The level of CPT I gene expression in the ventricles of the JVS mice was at least three- to sixfold that of normal mice as judged by reverse transcription-polymerase chain reaction (RT-PCR). When the JVS mice were treated with carnitine, CPT I gene expression was repressed to the level of normal mice. Therefore, the increased expression of the CPT I gene was associated with carnitine deficiency.