Determination of cobalamin and related compounds in foods.

The microbiological assay of total cobalamin (vitamin B12) by Lactobacillus delbrueckii subsp. lactis ATCC7830 is now used worldwide in food analysis because of its high sensitivity, low running cost, and no expensive instruments. It has been recently reported that some foods contain a substantial number of inactive corrinoid compounds, some of which are active in this bacterium. These results indicate that the microbiological method must be replaced with high-performance liquid chromatography or liquid chromatography/electrospray ionization-tandem mass spectrometry as there can specifically determine biologically active cobalamin. Nowadays, powerful tools, such as immunoaffinity columns, purify cobalamin simply and specifically. In this chapter, we summarized the determination methods of cobalamin and related compounds in foods. Various inactive corrinoids found in foods were also characterized.

A crucial role of plasma membrane-associated sialidase in the survival of human cancer cells.

Human plasma membrane-associated sialidase (NEU3), a key enzyme for ganglioside degradation, is markedly upregulated in human cancers, leading to apoptosis suppression. To define molecular mechanisms and the possible target for NEU3, its encoding gene was silenced by small interference RNA (siRNA) or overexpressed in human cells. NEU3 siRNA-induced apoptosis with no special stimuli in HeLa cells, accompanied with decreased Bcl-xL and increased mda7 and GM3 synthase mRNA levels, whereas overexpression resulted in the opposite. Carcinoma HT-29 and MCF-7 cells appeared to be similarly affected, but normal cell lines demonstrated no significant changes. NEU3 siRNA was found to inhibit and NEU3 overexpression to stimulate Ras activation with consequent influence on extracellular signal-regulated kinases and Akt. Ras activation by NEU3 was abrogated by PP2 (src inhibitor) or AG1478 (epidermal growth factor receptor (EGFR) inhibitor), and NEU3 actually enhanced EGF-stimulated tyrosine-phosphorylation of EGFR, suggesting that the upstream targets might be tyrosine kinases including src and EGFR, and the subsequent stimulation of Ras cascade leads to the inhibition of cell apoptosis. Glycolipid changes observed seemed to be one of the causes of the cell effects. NEU3 may thus be an essential gene for cancer cell survival and siRNAs targeting this protein could have utility for gene-based therapy of human cancers.

A structural study of calcium-binding equine lysozyme by two-dimensional 1H-NMR.

Since 1H-NMR spectra of the calcium bound form (holo) and the calcium free form (apo) of equine lysozyme have an overall similarity, the folded structure of apo equine lysozyme seems to be similar to the holo structure at 25 degrees C and pH 7.0, even at low ionic strengths except for subtle conformational change. However, calcium titration experiments showed that a number of resonances change by a slow exchange process. The changes saturated at one calcium ion per one lysozyme molecule, and no more change was observed by further addition of calcium ions. This shows that just one calcium ion binds to equine lysozyme. To make assignments for these changed proton resonances, two-dimensional 1H-NMR studies, correlated spectroscopy (COSY), two-dimensional homonuclear Hartmann-Hahn spectroscopy (HOHAHA) and nuclear Overhauser effect spectroscopy (NOESY) were carried out. A structural model of equine lysozyme based on the crystal structure of human lysozyme was estimated and used to assign some resonances in the aromatic and beta-sheet regions. It was possible to use some proton signals as a probe to determine the specific conformational change induced by calcium ions. The calcium binding constant KCa was estimated from calcium titration experiments in which changes in the proton signal were monitored. The log KCa value was found to be on the order of 6-7, which is in agreement with the calcium binding constant determined by fluorescence probes. This means that the protons are affected by specific calcium binding.

Cloning and sequencing of the murine Mxi1 cDNA.

The cloning and sequencing of the murine Mxi1 gene encoding Mxi1, one of Max-associated proteins, is described. Murine and human sequences showed 87.9% nucleotide (nt) and 90.3% amino acid (aa) sequence homology, whereas murine and zebra fish sequences showed 67.2% nt and 67.8% aa sequence homology.

Attention changes the peak latency of the visual gamma-band oscillation of the EEG.

To investigate the physiological role of visual gamma-band oscillation (GBO), we calculated the event-related dynamics of the EEG power-spectrum for paired visual stimuli (S1 and S2) with or without attention in 12 subjects. The visual stimuli elicited transient increases in the GBO power (around 40 Hz), which were maximal over the parietal area. The peak GBO increase appeared around 300 ms after stimulus onset, but its latency was shorter after S1 and longer after S2 under the 'with attention' than under the 'without attention' condition. This transient increase in the visual GBO is thought to reflect attention and to reset the activity of the visual system in preparation for a new stimulus.

The synchronization between brain areas under motor inhibition process in humans estimated by event-related EEG coherence.

To investigate the functional connection of brain areas under motor inhibition, the event-related coherence (ERCoh) of the electroencephalogram (EEG) was calculated for 10 subjects who were asked to perform a visual discrimination (GO/NO-GO) task. The subjects were instructed to push (GO) or not to push (NO-GO) a micro-switch according to different visual stimuli. Twenty-one-channel scalp EEGs were recorded and the surface Laplacians were calculated at F3, F4, C3, C4, P3 and P4 using the source derivation method. The time-courses of the inter- and intra-hemispheric coherence were calculated using the fast Fourier transform for each condition (GO or NO-GO), and were compared statistically between the two conditions. The results suggest that the ERCoh under the NO-GO condition consisted of two components; alpha band synchronization between bilateral frontal areas and theta band synchronization among bilateral frontal, central and parietal areas. It is likely that the former is related directly to the decision not to move, and the latter is related to the motor inhibition process.

Event-related dynamics of the gamma-band oscillation in the human brain: information processing during a GO/NOGO hand movement task.

To investigate the gamma band activity relating to the discrimination process and motor behavior in the human brain, the event-related dynamics of the EEG spectrum was calculated during the visual GO/NOGO hand movement task and a control task (the visual element of the GO/NOGO task only) in eight subjects. The subjects were instructed to push (GO) or not to push (NOGO) a microswitch according to different visual stimuli and 21-channel scalp EEGs were recorded. The time courses of the power spectra after the stimuli were calculated using the fast Fourier transform for each condition (GO, NOGO and the control task), and were compared statistically between the conditions. The results suggested that a high gamma band oscillation, occurring at the frontal and left parieto-occipital areas at around 90 ms after the stimuli, relates to the discrimination process. Under the GO condition, this oscillation continued until 140 ms, and a subsequent oscillation occurred over the motor areas at around 200 ms, which seemed to be related to the motor action. On the other hand, under the NOGO condition, a low gamma band oscillation occurred in the central area at around 230 ms, which seemed to be related to the inhibition process.

Altered expression levels of G protein subclass mRNAs in various seizure stages of the kindling model.

The expressions of mRNAs encoding G protein alpha subunits were analyzed in the cerebral cortex of amygdaloid kindled rats. A remarkable increase in Gsalpha mRNA were observed on the bilateral cerebral cortex at 24 h after the last generalized seizure and persisted 3 weeks on the unstimulated side. Gi2alpha mRNA level was also increased on the stimulated side at 24 h and persisted 3 weeks. These result suggest that dysfunction of Gs and Gi2 might relate to the basic mechanisms of seizure generation and the maintenance of epileptogenesis.

The time course of interhemispheric EEG coherence during a GO/NO-GO task in humans.

Event-related coherence of the EEG was calculated for 10 subjects performing a visual discrimination GO/NO-GO task. The subjects were instructed to push (GO) or not to push (NO-GO) a button according to visual stimuli. Twenty-one-channel scalp EEGs were recorded and the surface Laplacian was calculated using the source derivation method. The time courses of the coherence between F3 and F4, C3 and C4, and P3 and P4 were calculated using the fast Fourier transform for each task and were compared between conditions. Statistical analysis showed that coherence in the NO-GO condition became significantly higher than that in the GO condition between F3 and F4. The synchronization between bilateral dorsolateral frontal areas might therefore play an important role in the motor inhibition process.

MR in temporal lobe epilepsy: early childhood onset versus later onset.

PURPOSE: To study the relationship between the MR findings and the clinical features in temporal lobe epilepsy in childhood (less than 10 years of age). METHODS: MR studies were performed with a 1.5-T imager on 38 temporal lobe epilepsy patients receiving drug therapy at the psychiatric department. These patients were divided into two groups according to their age at onset (10 years or less, 11 years or more). The two groups were compared in terms of the MR findings and clinical features. RESULTS: The 11 younger-onset patients included 5 with a high-signal area attributed to mesial temporal sclerosis. Clinically, all of these 5 patients had a history of "complex" febrile convulsions, which sharply distinguished them from the older-onset group. CONCLUSION: The analysis suggests that complex febrile convulsions in infancy can be associated with high-signal areas on MR attributed to mesial temporal sclerosis.

Structural specificity for biological activity of trichostatin A, a specific inhibitor of mammalian cell cycle with potent differentiation-inducing activity in Friend leukemia cells.

Biological activities of four chemically synthesized trichostatin-related compounds, (R)-trichostatin A, (S)-trichostatin A, (R)-trichostatic acid, and (S)-trichostatic acid, were investigated. Assays of differentiation-inducing activity in Friend leukemia cells and G2-arresting activity in the cell cycle of normal rat fibroblast cells were used as monitoring systems for comparing the bioactivities of these compounds. The results clearly showed that both of the enantiomers of trichostatic acid had no activity in both the assay systems. In the case of (S)-trichostatin A, the antipode of naturally occurring trichostatin A, 50% effective concentrations were determined to be 50-70-fold higher than those of (R)-trichostatin A. The relationship between this ratio and the value of enantiomeric excess strongly suggests that (S)-trichostatin A is also biologically inactive. These results indicate that the absolute configuration and the presence of the hydroxamate group of trichostatin A are essential for its biological activity.

Anterior dislocation of the subtalar joint: a case report.

We report a very rare case of anterior dislocation of the subtalar joint. Forceful supination of the foot and dorsiflexion of the ankle was considered the cause of the injury in this case. Closed reduction was successful for the talocalcaneal component of subtalar joint, although surgery was subsequently performed because of the residual subluxation of the midtarsal joint including the talonavicular component of subtalar joint and the associated fracture of the lateral process of the talus. Satisfactory results were shown at three-year follow-up.

[A case of "interval" form of acute carbon monoxide poisoning--brain MRI and therapeutic effect of hyperbaric oxygenation].

A 43-year-old female who had suffered from paranoid reaction attempted suicide with city gas. She was admitted to an emergency hospital in a comatose state, regained consciousness the next day and was discharged 12 days after. However, on the 21st day after the poisoning, behavioral anomalies and staggering appeared. She was readmitted into another emergency hospital with the diagnosis of an "interval" form of acute carbon monoxide poisoning. She was performed hyperbaric oxygen therapy 42 times until the 53rd day after the accident. Nevertheless, her symptoms did not improve and remained in a state like an apallic syndrome. With little promise of improvement, she was moved to our hospital, and hyperbaric oxygen therapy further continued for 40 times which resulted in a remarkable recovery. Repeat EEGs were performed. On the 32nd day, EEG showed an abnormality consisting of generalized delta waves. On the 56th day, the abnormal EEG improved slightly with more alpha activity. EEG was normalized 126 days after. The X-ray CT scan on the 32nd day revealed low densities in the bilateral globus pallidus and the deep white matters adjacent to the frontal horns of the lateral ventricle and in the semioval centers. On the 57th day, these low density areas were depicted more clearly by the X-ray CT scan, which became less marked on the 73rd day. MRI was performed 3 times.(ABSTRACT TRUNCATED AT 250 WORDS)

[Evaluation of the clinical effect of ceftazidime in the field of obstetrics and gynecology].

Ceftazidime (CAZ) was administered to patients with obstetrical and gynecological infectious diseases and we studied its clinical effect: CAZ was administered to 24 patients at a dose of 2--4 g per day (twice a day) by drip infusion over a period of 3--11 days (total 6--32 g). The subjects included 19 cases of intrauterine infection, 4 of adnexitis, and 1 of infection of the external genital organs. The overall clinical results were excellent in 6 cases and good in 18 cases, the efficacy rate being 100%. For bacteriological study, 26 strains were isolated from 16 patients. These included Gram-positive bacteria (10 strains), Gram-negative bacteria (6 strains) and anaerobes (10 strains). After CAZ treatment, 4 strains (S. mitis 1, E. coli 1, P. anaerobius 1, B. melaninogenicus 1) were eliminated, but as to the other 22 strains, the results were unknown, because we could not examine microbiology after administration. No clinical side effect or abnormality in laboratory findings was observed.

[Renal artery embolism treated with a continuous selective intra-arterial urokinase infusion therapy: a case report].

A 61-year-old man with left renal artery embolus was treated with a continuous selective intra-arterial urokinase (UK) infusion therapy. UK was administered through a 5 Fr. catheter embedded into the occlusive segment of the artery selectively. Continuous low dose UK (15,000 units/hour) was infused following short-term ultra-high dose UK (480,000 units/60 min) infusion. A complete recanalization of the occluded artery was observed by day 7 and also no serious complications were noted during the treatment. Thus, a continuous selective intra-arterial UK infusion therapy seemed to be more effective than conventional methods, especially in patients with complete obstruction.

[Neoadjuvant intra-arterial chemotherapy based on chemosensitivity tests for locally invasive bladder cancer].

We investigated the clinical usefulness of individualization of chemotherapeutic regimen in neoadjuvant intra-arterial chemotherapy for locally invasive bladder cancer. Anticancer drugs were selected according to the results of an in vitro chemosensitivity test (collagen matrix assay or succinic dehydrogenase inhibition test). Nine patients with locally invasive bladder cancer received 1 to 4 courses of neoadjuvant intra-arterial chemotherapy, followed by radical cystectomy. Histopathological responses in the cystectomized specimens were grade 3 in 3 cases, grade 2 in 2, grade 1b in 2 and no response in 2. Pathologically, a complete response and downstaging were observed in 3 and 4 cases, respectively. Seven of the 9 patients were alive no evidence of disease with a mean follow-up period of 38.9 months, whereas 2 patients died of metastasis within 2 years. Six of the 7 patients who showed a complete response or down staging have been free of recurrence. These findings suggest that our chemotherapeutic strategy may improve the prognosis for locally invasive bladder cancer.

[A case of urachal abscess].

A 26-year-old male with urachal abscess was demonstrated. Ultrasonography, CT and MRI revealed that there was a mass at the supravesical region, which was connected to the umbilicus. However, a differential diagnosis as to whether the mass was a tumor or an abscess was difficult from these examinations. Thus we performed an open biopsy. A pathological examination revealed no malignant change in the biopsy specimen. On the other hand, the pathological diagnosis of a follicular tumor located at the roof of the urinary bladder was TCC G2, but we considered that, this change was a reactive hyperplasia with dysplasia resulting from chronic inflammation of urachal abscess. Thus, the complete excision of urachal abscess and partial cystectomy were performed. The serial sections of the bladder wall revealed no malignant changes. We reviewed similar cases reported in our country and discussed various factors causing urachal abscess, especially related to the infectious root.

[Chemosensitivity test on superficial urinary bladder cancer using the dye exclusion assay--model of intravesical chemotherapy].

A chemosensitivity test was carried out on superficial bladder cancers using the trypan blue dye exclusion assay for the purpose of screening chemosensitive drugs for intravesical chemotherapy. Transplantable murine bladder tumor cells (MBT-2) were incubated, in vitro, in the presence of adriamycin (4, 40, 400, 1000 micrograms/ml) as well as verapamil (3, 30, 100, 500 micrograms/ml) at 5% CO2, 37 degrees C for two hours. After cellular viabilities were calculated, MBT-2 cells were inoculated into the hind limbs of mice. The cellular viability was correlated well with the ratio of tumor appearance, tumor growth inhibition and prolongation of survival, and was dose dependent in the adriamycin treated groups. On the other hand, a reduction of cellular viability, tumor growth inhibition and prolongation of survival were seen in the high dose verapamil (100, 500 micrograms/ml) treated groups. Human superficial bladder cancer cells were incubated in the presence of adriamycin, 4'-0-tetrahydropyranyladriamycin, mitomycin C and pepleomycin (1000 micrograms/ml) and/or verapamil (500 micrograms/ml). The reduction rates of cellular viability markedly varied with the kind of anticancer drugs. A reduction of cellular viability of human tumor cells as well as MBT-2 cells was seen in the verapamil treated groups. This rapid and handy assay seems to be useful for the purpose of screening chemosensitive drugs for intravesical chemotherapy.

[Cytological changes in endocrine therapies of prostatic carcinoma].

We studied cytological changes by endocrine therapies of prostatic carcinoma in 31 cases. We originated a method of cytology, in which a prostatic fluid collecting catheter was used. This has been already reported in previous papers. By the therapies, carcinoma cells usually became edematous, swollen, squamous and finally keratinized and in the cytoplasm hematoxylin-stained granules often appeared. These findings were usually seen in two weeks after the onset of the endocrine therapy. But the degree of the changes differed among the cases and among the therapies. In some there was no response or only squamous metaplasia was found. Most of the carcinoma cells were viable. In these cases the therapies were thought to be ineffective and their mortality was high. On the contrary, in those with only keratinized cells, the therapies were thought to be effective and the mortality was low. We conclude that the cytological methods were useful for evaluating the therapies.

[Suicidal attempts in three postoperative patients with renal cancer after alpha interferon withdrawal].

We report 3 cases of suicide attempts in postoperative patients with renal cancer after alpha interferon withdrawal. In the first patient, depression occurred during interferon therapy, and remained after interferon withdrawal. A suicide attempt occurred 7 months after interferon withdrawal. In the second and third patients, depression did not occur during interferon therapy, but suicide attempts occurred 40 days and 7 months after interferon withdrawal, respectively. Depression does not always disappear after interferon is discontinued. Psychiatric supervision should be continued even more frequently after interferon withdrawal. The increased risk of psychiatric side effects due to interferon, as well as their severity, suggest that interferon should be administered with caution.