Paroxysmal dysarthria and ataxia after midbrain infarction.

The authors describe a patient who showed paroxysmal dysarthria and right-limb ataxia after midbrain infarction. SPECT imaging showed marked hypoperfusion in the left parietal lobe while the patient was having frequent paroxysmal attacks. After treatment with phenytoin, the symptoms and hypoperfusion in SPECT imaging improved. The authors conclude that dysfunction of the cerebellothalamocortical pathway after midbrain infarction may cause paroxysmal dysarthria and ataxia.

Targeted down-regulation of MLL-AF9 with antisense oligodeoxyribonucleotide reduces the expression of the HOXA7 and -A10 genes and induces apoptosis in a human leukemia cell line, THP-1.

The MLL gene is frequently rearranged in leukemias, and MLL chimeric proteins generated by chromosomal translocations play crucial roles in leukemogenesis. Targets of murine Mll include HOX proteins that regulate body pattern formation and hematopoiesis. However, it is not known whether or not the MLL chimeric proteins regulate the HOX gene expression in human leukemia. To address this issue, THP-1 cells, a human leukemia cell line expressing MLL-AF9, were treated with antisense oligodeoxyribonucleotide (ODN) complementary to the coding sequence of the MLL-AF9 junction. Down-regulation of the MLL-AF9 transcript was accompanied by the reduced expression of the HOXA7 and -A10 genes, but not of the HOXA2, -A4, -A5, and -A9 genes. The number of viable cells cultured with 20 microM antisense ODN for 5 days was 10-fold lower than that of the sense ODN-treated control. And the number of the annexin V-/propidium iodide- apoptotic cells in the antisense ODN-treated cells after 3 days of culture was two-fold higher than that in the control. Staining of the antisense ODN-treated cells with Hoechst 33258 showed the morphology characteristic to apoptosis. These results indicate that MLL-AF9 regulates the expression of the selected HOX genes as well as prevents the leukemic cells from apoptosis.

Design, synthesis, and biological evaluation of anti-HIV double-drugs. conjugates of HIV protease inhibitors with a reverse transcriptase inhibitor through spontaneously cleavable linkers.

Based on the prodrug concept as well as the combination of two different classes of anti-HIV agents, we designed and synthesized a series of anti-HIV double-drugs consisting of HIV protease inhibitors conjugated with a nucleoside reverse transcriptase inhibitor in an effort to enhance the antiviral activity. For the conjugation, a series of linkers that conjoins the two different classes of inhibitors has been investigated. Double-drugs using a succinyl amino acid linker were shown to release the parent drugs via spontaneous imide formation at a faster rate compared to compounds using a glutaryl amino acid linker, as expected from the energetically favorable cyclization to the five-membered ring. Among the double-drugs, KNI-1039 (3b) with a glutarylglycine linker exhibited extremely potent anti-HIV activity compared with that of the individual components. Double-drug 3b was relatively stable in culture medium, whereas it regenerated active species in cell homogenate. These results suggested that the synergistic enhancement of anti-HIV activities of 3b may be due to their ability to penetrate into the target cell and subsequent regeneration of two different classes of anti-HIV agents in the cytoplasm.

Post-operative adjuvant chemotherapy for colorectal cancer with 5-fluorouracil (5-FU) infusion combined with 1-hexylcarbamoyl-5-fluorouracil (HCFU) oral administration after curative resection.

BACKGROUND: Although surgical resectability is an important prognostic factor, recurrences are commonly noted in advanced colorectal cancer patients, even after apparently curative surgery. Since such recurrences cannot be cured, better adjuvant chemotherapies are urgently required. PATIENTS AND METHODS: We studied the effect of post-operative chemotherapy using oral administration of 1-hexylcarbamoyl-5-fluorouracil (HCFU) with 5-fluorouracil (5-FU) infusion for curatively-resected Stage IIIa and IIIb colorectal cancers. This study was prospectively randomized and controlled and 314 (97.8%) out of 321 patients were determined to be candidates for statistical assessment. Group A and Group B received 5-FU intravenous injection at, respectively, 333 mg/m2 and 1000 mg/m2 body surface area/24 hours continuously for 72 hours beginning on post-operative day 0 and day 6, with oral HCFU 300 mg daily for 52 weeks beginning 2 weeks after surgery. RESULTS: There were no differences in overall 5-year survival or disease-free survival between Group A and Group B. A retrospective subset analysis. however, suggested that the protocol of Group B tended to yield better 5-year survival (68.3%) for rectal cancer than that of Group A (58.8%). CONCLUSION: Inductive therapy with high-dose 5-FU in combination with oral HCFU appears to be beneficial as adjuvant chemotherapy for advanced rectal cancer with lymph node metastasis.

cDNA cloning and expression of mutant catalase from the hypocatalasemic mouse: comparison with the acatalasemic mutant.

Mutant catalase cDNAs from the hypocatalasemic and acatalasemic mice were cloned and expressed in bacteria. A novel missense mutation, Asp (AAT) to Ser (AGT), was identified at amino acid position 439 of the hypocatalasemic catalase. Analysis of recombinant catalase mutants revealed that the mutation is responsible for the reduced activity of hypocatalasemic catalase and the unstable tetrameric structure of acatalasemic catalase was also suggested.

Amino-acid replacements in an internal region of tropomyosin alter the properties of the entire molecule.

Two isoforms of lobster muscle tropomyosin, a fast muscle type, fTm, and a slow muscle type, sTm1, are identical except for 15 residues within the region of amino acids 39-80, which corresponds to exon 2 of the tropomyosin genes of many phyla. Although the difference in the sequence does not include the terminal regions, the two isoforms are extremely different in viscosity, which is a good measure of the head-to-tail interaction strength and should be dependent on the conformation of the terminal 7-9 residues. To determine the influence of amino-acid replacements in the internal region on the overall conformation and the functional properties of the molecule, we compared the physical properties of the two isoforms and their interactions with other proteins, such as actin and myosin subfragment 1 (S1). Limited proteolysis by trypsin and chymotrypsin showed that sTm1 is more susceptible than fTm at the sites outside the region with the replaced residues. Compared with fTm, sTm1 showed higher viscosity, had a higher actin affinity, and inhibited acto-S1 ATPase to a greater extent. Finally, the binding isotherm of S1-ADP to actin-sTm1 is less sigmoidal than that to actin-fTm. These results indicate that the amino-acid replacements in the internal region alter the conformation and the physical properties of the entire molecule as well as its interactions with actin and myosin.

Novel SH3 protein encoded by the AF3p21 gene is fused to the mixed lineage leukemia protein in a therapy-related leukemia with t(3;11) (p21;q23).

The mixed lineage leukemia (MLL) gene located at chromosome band 11q23 is frequently rearranged in patients with therapy-related acute monocytic leukemia who received topoisomerase II inhibitors. We have identified a novel fusion partner of MLL (FAB M5b) in a patient who developed t-AML 9 years after treatment for acute lymphoblastic leukemia (ALL). The leukemic cells had a sole karyotypic abnormality of t(3;11) (p21;q23). Screening of a genomic DNA library, prepared from leukemic cell DNA, identified rearranged clones composed of MLL and a novel gene on chromosome 3p21 (AF3p21). The AF3p21 gene encodes a protein of 722 amino acids, which contains an Src homology 3 (SH3) domain, a proline-rich domain, and a bipartite nuclear localizing signal (NLS). RNA analysis demonstrated that exon 6 of the MLL gene fused to exon 2 of the AF3p21 gene. The resulting chimeric protein consists of AT-hooks, methyltransferase, and transcription repressor domains of MLL in addition to the AF3p21 proline-rich domain and NLS but not the AF3p21 SH3 domain.

Randomized controlled trial of 5-fluorouracil (5-FU) infusion combined with 1-hexylcarbamoyl-5-fluorouracil (HCFU) oral administration and HCFU alone as postoperative adjuvant chemotherapy for colorectal cancer.

BACKGROUND: Although surgical resectability is an important prognostic factor, recurrences are commonly noted in advanced colorectal cancer patients, even after apparently curative surgery. Because such recurrences cannot be cured, better adjuvant chemotherapies are urgently required. PATIENTS AND METHODS: We studied the effect of postoperative chemotherapy using 1-hexylcarbamoyl-5-fluorouracil (HCFU) oral administration with or without 5-fluorouracil (5-FU) infusion for curatively resected Stage II and III colorectal cancer. This study was prospectively randomized and controlled and 303 (95.6%) of 316 patients were determined to be candidates for statistical assessment. Group A received oral HCFU, 300 mg daily for 52 weeks beginning 2 weeks after surgery. Group B also received 5-FU intravenous injection, 333 mg/m2 body surface area/24 hours continuously for 72 hours beginning on postoperative day 0 and 6. RESULTS: There were no differences in overall 5-year survival or disease-free survival between Groups A and B. Group B had better 5-year disease-free survival (47.6%) than Group A (42.9%) (p = 0.062) and significantly prolonged interval from surgery to recurrence (p = 0.003) for patients with lymph node metastasis. In contrast, group B had significantly shortened 5-year disease-free survival (p = 0.010) and increased recurrence rate in patients without lymph node metastasis. CONCLUSION: Inductive therapy with 5-FU in combination with oral HCFU is beneficial as adjuvant chemotherapy for advanced colorectal cancer with lymph node metastasis.

Peroxisome-proliferator regulates key enzymes of the tryptophan-NAD+ pathway.

Structually diverse peroxisome-proliferators (PPs) were investigated regarding their effects on NAD+ level and two key enzyme activities in the tryptophan (Trp)-NAD+ pathway in the liver of rats (Sprague-Dawley male) fed PP-containing diets freely for 2 weeks. All PPs, except for thyroxine, significantly increased hepatic NAD+ level in concert with hepatic hypertrophy. Activity of quinolinate phosphoribosyltransferase (QAPRTase), one of the key enzymes in the Trp-NAD+ pathway, was increased by the PPs which caused significant increase in the hepatic NAD+. On the other hand, alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSDase), another key enzyme in the Trp-NAD+ pathway, was drastically inhibited by all PPs except for linolenic acid, which was only slightly inhibitory. Most PPs investigated activated peroxisomal marker enzymes such as palmitoyl-CoA oxidase, catalase, and PPAR-alpha(peroxisome-proliferator activated receptor-alpha)-dependent enzymes, such as malic enzyme and l-3-glycerophosphate dehydrogenase. NAD+ was also increased in the rat hepatocytes cultured in the medium supplemented with PPs. These data suggested that regulation of the key enzymes in the Trp-NAD+ pathway was associated with PPAR-alpha directly or indirectly, and as a consequence the hepatic NAD+ was increased by PPs.

Cloning of tropomyosins from lobster (Homarus americanus) striated muscles: fast and slow isoforms may be generated from the same transcript.

Complementary DNAs encoding fibre-type-specific isoforms of tropomyosin (Tm) have been isolated from lobster (Homarus americanus) striated muscle expression libraries made from poly(A)+ RNA purified from deep abdominal (fast-type) and crusher-claw closer (slow-type) muscles. A cDNA of slow-muscle Tm (sTm1), containing a complete open reading frame (ORF) and portions of the 5' and 3' untranslated regions (UTRs), encodes a protein of 284 amino acid residues with a predicted mass of 32,950, assuming acetylation of the amino terminus. The nucleotide sequence of a fast-muscle tropomyosin (fTm cDNA), which includes the entire ORF and part of the 3' UTR, is identical to that of sTm1 cDNA, except in the region encoding amino acid residues 39-80 (equivalent to exon 2 of mammalian and Drosophila muscle tropomyosin genes). The deduced amino acid sequences, which display the heptameric repeats of nonpolar and charged amino acids characteristic of alpha-helical coiled-coils, are highly homologous to tropomyosins from rabbit, Drosophila, and shrimp (57% to 99% identities, depending on species). Northern blot analysis showed that two transcripts (1.1 and 2.1 kb) are present in both fibre types. Mass spectrometry indicated that fast muscle contains one major isoform (fTm: 32,903), while slow muscle contains two major isoforms (sTm1 and sTm2: 32,950 and 32,884 respectively). Both Tm preparations contained minor species with a mass of about 32,830. Sequences of peptides derived from purified slow and fast Tms were identical to the deduced amino acid sequences of the sTm1 and fTm cDNAs, respectively, except in the C-terminal region of fTm. The difference in mass between that predicted by the deduced sequence (32,880) and that measured by mass spectrometry (32,903) suggests that fTm is posttranslationally modified, in addition to acetylation of the N-terminal methionine. These data are consistent with the hypothesis that the fTm and sTm1 are generated by alternative splicing of two mutually-exclusive exons near the 5' end of the same gene.

Production and crystallization of lobster muscle tropomyosin expressed in Sf9 cells.

A new form of muscle tropomyosin crystal has been obtained, by employing new strategies in protein preparation and crystallization. Non-polymerizable tropomyosin was prepared by removing 11 amino acids at the C-terminus. The truncated tropomyosin was expressed in Sf9 insect cells by use of the baculovirus-based expression system, to obtain highly homogeneous protein preparations. By routinely monitoring homogeneity by mass spectrometry, we found that the homogeneity played a key role in obtaining good crystals. The crystal quality was also dependent on isoforms; the crystals raised from a slow muscle-specific isoform diffracted to a higher resolution, compared with a fast muscle-specific counterpart. For crystallization, a high concentration of organic solvent was used as the precipitant; in the presence of 35% DMSO, tetragonal crystals were formed, which belong to space group P4(3)(1)2(1)2 with cell constants of a=b=105.6 angstrom, c=506.9 angstrom. The crystals gave rise to reflections the intensities of which were characteristically determined by the transform of alpha-helical coiled-coil. Thus in the region of 10-5.5 angstrom resolut along the c*-axis, the reflections were weak. For accurate measurement of these reflection intensities, beam-line ID2 in ESRF Grenoble was advantageous owing to the high brilliance and a low background. There the crystals diffracted to beyond 3.0 A along the c*-axis, whereas along the a*-b*-plane reflections were limited to 6.6 angstrom. Data analysis is under way on a data set from a PtCl4 derivative.

Experimental study of combined hyperthermic and photodynamic therapy on carcinoma in the mouse.

PURPOSE: This study investigates the cytotoxic effect of photodynamic therapy using high-power laser irradiation on cancer cells. MATERIALS AND METHODS: High- or low-power irradiation from a pulsed Nd:YAG dye laser with or without a photosensitizer was administered to an NR-S1 carcinoma in the mouse dorsum. RESULTS: Photodynamic therapy with high-power laser irradiation yielded better results than conventional photodynamic therapy or hyperthermia with high-power laser irradiation. CONCLUSION: Photodynamic therapy with high-power laser irradiation is more effective because it generates both a hyperthermic and a photodynamic effect.

Perceptual changes accompanying controlled blocking of cutaneous afferents are related to somatosensory evoked potentials.

We examined the effect of local anaesthesia of the skin on somatosensory evoked potentials (SEPs) and psychophysical magnitude estimates for a constant intensity air-puff stimulation (7 kg.cm-2) in 23 normal subjects. Before and after intradermal injection (0.1 ml of 1% xylocain) to the tip of the right index finger, magnitude estimations for the four modalities of skin sensation (touch, pressure, pain and vibration), and SEPs were successively examined at 4-10 min interval until complete sensory and SEP recovery. The time course of sensory recovery was similar for the first three modalities and vibration sense was little affected by local anaesthesia. Immediately after anaesthesia, SEPs either abolished or decreased in amplitude. Thereafter amplitudes increased and peak latencies decreased with the elapsed time. Comparison of the regression lines for the amplitude of P45-N60 component or the time-integrals of N20 and N35 components with that of psychophysical estimates revealed steeper slopes for psychophysical data: 0.4-0.7 for neural versus 1.4-1.7 for psychophysical data. These relations between SEPs and psychophysics following local anaesthesia bear a close parallel to those observed in our previous studies in a normal condition with increasing stimulus intensity. The SEP latencies for N20, P27 and N35 components were better correlated with recovery from anaesthesia. Direct comparisons of SEP measures with subjective magnitudes produced significant correlations for the three modalities of sensation in which again latencies are better correlated than amplitides or time-integrals.

Thalamic atrophy following cerebral infarction in the territory of the middle cerebral artery.

We investigated shrinkage of the ipsilateral thalamus following infarction in the territory of the middle cerebral artery in 33 patients who were admitted less than or equal to 2 days after the stroke and who were followed by computed tomography for greater than 1 year with no recurrences. The thalamic area was measured on the computed tomograms, and the ratio of the ipsilateral area to the contralateral area was calculated. All values were compared with values from the initial computed tomogram taken less than or equal to 2 days after the stroke. The values of the ratio on follow-up computed tomograms decreased gradually in 15 patients. In these cases, the area of the ipsilateral thalamus was significantly reduced after 1 year (p less than 0.01) and marked atrophy was observed. These results demonstrate the significance of remote changes over a long period of time after focal cerebral infarction.

Posteromedial hypothalamotomy in the treatment of violent, aggressive behaviour.

Although emotion in the human is largely modified by the frontal association areas (software) and may better be called affect, it is still very much influenced by the balance of the ergotropic and the trophotropic circuits in the prosencephalon (hardware) especially in patients with organic brain lesions. Violent, aggressive, restless behaviours or rage can be regarded as an unbalanced state of these two circuits with dominance of the ergotropic circuit. In order to restore the balance of these two circuits, small stereotactic lesions were made in the ergotropic portion of the posterior hypothalamus (posteromedial hypothalamotomy) with good results in the follow-up of 10-25 years. Postoperatively there was no disturbance in endocrine activities and growth.

[Effects of intravenous alimentation on adjuvant chemotherapy--an experimental study on the distribution of 5-FU after injection of tegafur (2)].

The stomach, small and large intestines, heart, lungs, bone and kidneys were removed from 48 Sato lung cancer-bearing rats used in the previous experiments and given 90 mg/kg of tegafur (FT-207) by single intravenous administration and tissue 5-FU and FT-207 concentrations were measured. FT-207 concentration in the alimentary canal was somewhat lower than the blood concentration, but both were lowered in parallel. 5-FU concentration in the stomach and large intestines showed virtually identical changes in both IVH and PO groups, but IVH group tended to have higher concentration. IVH group showed higher values than PO group anytime, particularly in the large intestines. A reduction of the side effects on the digestive system via intravenous alimentation was thought due to the elimination of mechanical stimulation via a cessation of oral feeding. 5-FU concentration in the bone was highest in PO group at six hours after administration and blood concentration changes were parallel, but there was virtually no change in IVH group. Maximum values were found one hour after administration and slowly declined thereafter; at 24 hrs the values were 0.059 +/- 0.013 microgram/g, relatively high compared to the PO group at 0.041 +/- 0.022 microgram/g. In the present study under intravenous alimentation, the concentration changes were slight in spite of 5-FU maximum concentration being lower than that by oral feeding and the long-term high concentration which was maintained; this is thought to be a disadvantageous action with regard to the bone marrow. FT-207 concentration in the kidney, heart and lungs was the same as that for the blood, with a gradual reduction in IVH group. 5-FU concentration was the same for the kidneys and IVH group quickly reached to the high levels compared to PO group with only slight changes thereafter. Effects of continuous water load might be involved but not clear.

[Effects of intravenous feeding on adjuvant chemotherapy--an experimental study on the distribution of 5-FU after injection of tegafur (1)].

Forty-eight male Donryu rats inoculated with Sato lung cancer were used to experimentally determine the effects of intravenous feeding on the concentrations of the chemotherapeutic agents FT-207 and 5-FU in the blood, as well as in the liver and tumorigenic tissue. Following FT-207 administration, the blood, tumor and liver tissue levels were lower than in the IVH group (oral administration). The liver 5-FU concentration, at 0.10 +/- 0.02 microgram/g, was significantly higher in the intravenous feeding group than in the p.o. group (0.05 +/- 0.01 micrograms/g). The 5-FU blood concentration rose quickly, reaching 0.051 +/- 0.013 micrograms/ml and 0.035 +/- 0.004 micrograms/ml at 9 and 12 hours, respectively, following treatment. This was significantly higher than in the p.o. group, which showed corresponding levels of 0.031 +/- 0.004 microgram/ml and 0.022 +/- 0.002 microgram/ml, respectively. The increase in the 5-FU level within the tumor was markedly high in the IVH group compared to the p.o. group, and it peaked at 9 hours following administration. The concentration in the IVH group was thus higher than in the p.o. group at any given time. At 24 hours after treatment, the IVH group level was 0.35 +/- 0.09 microgram/g, against 0.27 +/- 0.05 microgram/g in the p.o. group. The blood concentration of 5-FU following intravenous feeding maintained a high value for a long time, and the 5-FU tumor concentration also remained at a high level. The intravenous route was therefore considered to be advantageous for antitumor chemotherapy.