Interaction between NUDT15 and ABCC4 variants enhances intolerability of 6-mercaptopurine in Japanese patients with childhood acute lymphoblastic leukemia.

6-Mercaptopurine (6-MP) is a main component of childhood acute lymphoblastic leukemia (ALL) treatment. Some candidate gene variants are associated with its toxicities, but the major variants and effects of combined variants remain unclear. We used Cox regression analysis to evaluate the time-dependent association between candidate variants and the cumulative incidence of 6-MP intolerability in 95 Japanese patients. The major risk factors for severe leukopenia were ABCC4 rs3765534, NUDT15 rs116855232 and rs186364861 in multi-covariate analysis (P<0.05). NUDT15 intermediate activity variant, that is, heterozygous rs116855232 or rs186364861 variant, and the ABCC4 rs3765534 variant showed leukopenia more frequently than either variant alone. All patients with both the intermediate activity NUDT15 variant and the ABCC4 rs3765534 variant suffered from leukopenia, and 57.1% patients required 50% protocol dose by day 168. These data indicate that NUDT15 and ABCC4 are major factors for 6-MP intolerability and that the interaction between these variants enhances intolerability to 6-MP.

Multidrug resistance protein 4 (MRP4) polymorphisms impact the 6-mercaptopurine dose tolerance during maintenance therapy in Japanese childhood acute lymphoblastic leukemia.

Multidrug resistance protein 4 (MRP4) is involved in the efflux of nucleoside derivatives and has a role in the determination of drug sensitivity. We investigated the relationship between MRP4 genetic polymorphisms and doses of the 6-mercaptopurine (6-MP) and methotrexate. Further, we evaluated the frequency of therapeutic interruption during maintenance therapy in Japanese children with acute lymphoblastic leukemia (ALL). Ninety-four patients received an initial 6-MP dose in the range of 30-50 mg m(-2) in this analysis. Patients with homozygous variant allele in any of MRP4 G2269A, C912A and G559T required high frequency of 6-MP dose reduction compared with non-homozygous individuals. Average 6-MP dose for patients with homozygous variant allele on either MRP4 or inosine triphosphate pyrophosphatase was significantly lower than that for patients with non-homozygous variant allele during maintenance therapy (30.5 versus 40.0 mg m(-2), P=0.024). Therefore, MRP4 genotyping may be useful for personalizing the therapeutic dose of 6-MP during the ALL maintenance therapy in Japanese.

Saposin-C from bovine spleen; complete amino acid sequence and relation between the structure and its biological activity.

Saposin-C, a small acidic glycoprotein that can activate glucosylceramide-beta-glucosidase, has been isolated from bovine spleen. The complete amino acid sequence of bovine saposin-C was determined by Edman degradation of the purified protein and its fragmented peptides. It contains 80 amino acids, one carbohydrate chain attached to a single asparagine residue and six cysteine residues in oxidized form. The sequence of bovine saposin-C is 76 and 65% identical with the sequences of saposin-C from human spleen and guinea pig liver, respectively. Hydropathy profiles of the sequence of saposin-C from three species were similar despite the significant residue substitutions. Bovine saposin-C had a stronger effect in stimulating bovine beta-glucosidase compared to human saposin-C. However, the effect of human saposin-C in stimulating human enzyme was stronger than that of bovine saposin-C. The region around residue 35, which is next to the extremely hydrophilic region, seems to be important to produce an interaction with the enzyme.

Further studies on D-3-aminoisobutyrate-pyruvate aminotransferase.

D-3-Aminoisobutyrate-pyruvate aminotransferase (EC 2.6.1.40, D-BAIB aminotransferase) participates in the metabolism of thymine. Recently we purified this enzyme from rat liver. We have studied D-BAIB aminotransferase further to clarify its physiological function. Among our findings were the following. (1) The enzyme activity was widely distributed in the organs of guinea pigs and rats. The kidney, liver, and lung showed high specific activities. (2) Using the livers of six vertebrates, differences between species were studied. Activity was detected in all species, the human liver showing the lowest activity among them. (3) Developmental study using rat liver showed that the activity was low at birth, increased sharply thereafter for 10 days, and then subsequently declined to the adult level. (4) Intraperitoneal injection of BAIB and beta-alanine in rats was performed to determine whether they induce activity of this aminotransferase. Only BAIB increased the activity of the aminotransferase in the liver significantly. (5) Subcellular distribution study of this aminotransferase in rat liver revealed that it is a mitochondrial enzyme.

Augmentation of megakaryocytopoiesis within the hematopoietic microenvironment of human granulocyte colony-stimulating factor transgenic mice.

OBJECTIVE: Megakaryocytopoiesis was dramatically augmented in human granulocyte colony-stimulating factor transgenic mice (G-Tg) compared to littermates. We examined the characteristics of megakaryocytes and megakaryocyte progenitor cells in these mice. MATERIALS AND METHODS: The numbers of colony-forming unit megakaryocytes (CFU-MK) and megakaryocytes in hematopoietic organs were counted. The megakaryocytes of G-Tg were examined ultrastructurally, and bone marrow transplantation studies using congenic G-Tg (Ly5.2) and C57BL/6 (Ly5.1) were performed. The number of day-14 colony-forming unit spleen (CFU-S) that contained megakaryocytes in [Ly5.1 > G-Tg] and [G-Tg > Ly5.1] mice also was counted. RESULTS: The number of CFU-MK increased markedly in the spleen, bone marrow, and peripheral blood. The number of megakaryocytes in the spleen and bone marrow also were increased in G-Tg mice. Ultrastructural analyses revealed that megakaryocytes in G-Tg mice were immature. Bone marrow transplantation studies of [Ly5.1 > G-Tg] mice resulted in a significantly increased number of megakaryocytes compared to [G-Tg > Ly5.1] mice. The number of day-14 CFU-S that contained megakaryocytes was increased markedly in [Ly5.1 > G-Tg] mice compared to [G-Tg > Ly5.1] mice. In vitro differentiation of megakaryocytes in [Ly5.1 > G-Tg] mice was induced by interleukin-11 and thrombopoietin. CONCLUSION: The results showed that the hematopoietic marrow microenvironment of G-Tg is important in augmenting megakaryocytopoiesis. [Ly5.1 > G-Tg] mice are potentially useful as a source of murine megakaryocytes and their progenitors.

Inhibitory mechanisms of H(+)-ATPase inhibitor bafilomycin A1 and carbonic anhydrase II inhibitor acetazolamide on experimental bone resorption.

The effects of the vacuolar-type H(+)-ATPase inhibitor bafilomycin A1 (baf.A1) and the carbonic anhydrase II inhibitor acetazolamide (AZ) on bone resorption and procathepsin L secretion of rat osteoclasts were investigated using the bone slice assay method, pit formation test. Baf.A1 completely suppressed osteoclastic bone resorption stimulated by parathyroid hormone (PTH), but did not affect procathepsin L secretion, while AZ suppressed both bone resorption and procathepsin L secretion. These findings suggest that bone resorption by procathepsin L secretion and its processing are regulated by proton production and proton secretion.

Distribution of prosaposin-like immunoreactivity in rat brain.

Prosaposin is the precursor for saposins A, B, C, and D, which are small lysosomal proteins required for the hydrolysis of sphingolipids by specific lysosomal hydrolases. With a monospecific anti-saposin C antibody, which cross-reacts with prosaposin but not with saposin A, B, or D, the present immunoblot experiments showed that the rat brain expresses an unprocessed approximately 72 kDa protein (possibly prosaposin) and little saposin C. Regional analysis demonstrated that prosaposin is abundant in the brainstem, hypothalamus, cerebellum, striatum, and hippocampus, and less abundant in the cerebral cortex. Consistent with this finding, prosaposin-like immunoreactive neurons and fibers as revealed by immunohistochemistry were observed frequently in subcortical regions. The medial septum, diagonal bands, basal nucleus of Meynert, ventral striatum, medial habenular nucleus, and motor nuclei of cranial nerve had significant numbers of immunoreactive neurons. There were also nerve fibers with prosaposin-like immunoreactivity in several projection fields of the above nuclei. Other brain areas that contained prosaposin-like immunoreactive neurons and/or processes were: several brain nuclei (nucleus caudate putamen, globus pallidus, substantia nigra, red nucleus) constituting the so-called extrapyramidal system, reticular thalamic nucleus, entopeduncular nucleus, mammillary nuclei, auditory relay nuclei, cerebellum, sensory cranial nerve nuclei, and the reticular formation. The distribution pattern of prosaposin is apparently different from that of other neuroactive substances so far examined, and thus prosaposin may be involved in novel central events.

DNA analysis in hereditary dentatorubral-pallidoluysian atrophy: correlation between CAG repeat length and phenotypic variation and the molecular basis of anticipation.

Hereditary dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disease with variable clinical phenotypes. Progressive ataxia, choreoathetosis, and dementia are the main clinical features of adult-onset cases, whereas the main feature in juvenile-onset DRPLA is progressive myoclonus epilepsy. Earlier onset is apparent in successive generations (anticipation). The molecular abnormality underlying DRPLA is an expanded, unstable CAG trinucleotide repeat on chromosome 12p. We analyzed 71 DNA samples obtained from 12 Japanese DRPLA pedigrees that included 38 affected individuals. Normal alleles had 7 to 23 repeats, DRPLA alleles 53 to 88 repeats. DRPLA alleles also were detected in five asymptomatic family members. Patients with juvenile onset had significantly larger repeats than did those with adult onset, and there was a significant negative correlation between CAG repeat length and age at onset. In 80% of the paternal transmissions, there was an increase of more than five repeats, whereas all the maternal transmissions showed either a decrease or an increase of fewer than five repeats. There was a significant correlation between father-child differences in repeat length and differences in age at onset. The analysis of CAG repeat length is a reliable diagnostic test for DRPLA and is of value for the presymptomatic detection of individuals at risk. The expansion of CAG repeats is important in phenotypic variation and anticipation. In addition, the sex of the transmitting parent has a significant effect on the molecular mechanism of anticipation.

Experimental intimal thickening studies using the photochemically induced thrombosis model in the guinea-pig femoral artery.

We have used the photochemically induced thrombosis model to study intimal thickening in the guinea-pig femoral artery. The femoral artery was occluded by a combination of rose bengal and green light which caused endothelial cell damage followed by formation of platelet rich thrombus at the site of endothelial damage. Thrombolysis was then achieved by administration of tissue-type plasminogen activator. Intimal thickening in the femoral arteries was histologically measured at 0, 1, 3, 6 and 9 weeks after the treatment. The neointimal areas gradually increased until 3 weeks and then remained unchanged up to 9 weeks. Cells in the neointima were identified as smooth muscle cells by immunohistochemical staining with an actin-specific antibody, HHF35. Cell proliferation in the media begun within 48 h after the thrombolysis and bromodeoxyuridine labeled cells appeared to migrate to the intima within 1 week after the thrombolysis. Administration of an angiotensin converting enzyme inhibitor, cilazapril (30 mg/kg/day, p.o.) for 3 weeks, suppressed intimal thickening and decreased the medial area in the femoral artery. These observations suggest that in this model cell proliferation and migration characteristics of pathological intimal thickening occur and this model is useful for investigating the effect of pharmacological preparations on intimal thickening.

HLA-D clusters associated with DR4 in the Japanese population.

A study of HLA-D clusters associated with DR4 was performed in the Japanese population. These clusters consist of DYT, DKT2, DB3, and Dw4. Forty-two Japanese typed as DR4 were investigated, and it was found that 17 (40.5%) were DYT, 7 (16.7%) DKT2, 7 (16.7%) DB3, and 4 (9.5%) Dw4.

In vitro and in vivo calcification of vascular bioprostheses.

Efficacy of different chemical treatments on calcification of vascular graft in vitro and in vivo was studied. Culture medium-filled rat aortas were separately treated in 0.2% glutaraldehyde and epoxy compound, and photooxidized in 0.01% methylene blue for a shorter period (group 1). Another group of rat aortas were separately treated in the same chemicals for a longer period (group 2). All fresh and treated aortas of both groups were cultured for 21 days in an organ culture medium and implanted (except for group 1) in weanling rats for five months. Histology and immunohistochemistry revealed that differently treated aortas of group 1 grow and calcify, and the smooth muscle cells between elastin fibers are the primary site of calcium deposition. In contrast, differently treated aortas of group 2 neither grew, nor did calcify in the medium except the epoxy compound cross-linked aorta of group 2 which did not grow but did calcify. Untreated aorta did not calcify. All fresh and differently treated aortic homografts calcified severely in rats. Our whole arterial segment-calcification system would be useful for analyzing the molecular and cellular mechanisms of both bioprosthetic and atherosclerotic calcification of vascular graft. New anticalcification technique is the only hope for better outcome of future vascular bioprostheses.

Human beta-defensin-2 induction in Helicobacter pylori-infected gastric mucosal tissues: antimicrobial effect of overexpression.

The objective of this study was to understand more of the innate immune response to Helicobacter pylori by determining the expression of human beta-defensin-2 (hBD-2) in various gastric mucosal tissues and MKN45 gastric cancer cells with or without H. pylori. Semi-quantitative TaqMan RT-PCR and immunohistochemistry were carried out. The antimicrobial effects of a transfected hBD-2 gene against H. pylori were also evaluated. The results showed that hBD-2 was expressed in inflamed gastric mucosal tissues with H. pylori infection, but not in the absence of H. pylori infection. Expression was also detected in gastric cancers in patients with H. pylori infection. Expression was induced in the MKN45 gastric cancer cell line by H. pylori in a manner dependent on the abundance of bacteria. hBD-2-transfected 3T3J2-1 cells secreted hBD-2 protein into the culture medium and this protein inhibited growth of H. pylori completely. The results suggest that hBD-2 may be involved in the pathophysiology of H. pylori-induced gastritis.

Inhibitory effect of methylene blue-induced photooxidation on intimal thickening of vein graft.

BACKGROUND: We have previously speculated that methylene blue-induced photooxidation of adventitial surface for 5 minutes can completely inhibit the intimal and medial growth of surgically prepared saphenous vein in vitro. In this study, inhibitory effect of methylene blue-induced photooxidation on intimal thickening of vein graft in vivo was investigated. METHODS: Jugular vein grafts were photooxidized in 0.01% methylene blue solution for 5 minutes, and interposed into arterial circulation for 4 weeks in rabbits. Vein grafts were studied by morphometry and immunohistochemistry. RESULTS: The intimal thickening of photooxidized vein grafts were suppressed significantly compared with those in the nonphotooxidized group. Proliferated cell nuclear antigen (PCNA) index (total PCNA-positive cells/total cell count x 100%) of vein graft was significantly higher in the nonphotooxidized group than those in the photooxidized group. CONCLUSIONS: Methylene blue-induced photooxidation is effective in the inhibition of intimal thickening of vein graft interposed in the arterial circulation for 4 weeks in vivo.

Percutaneous microexplosion nephrolithotripsy.

We developed the technique of microexplosion for the destruction of bladder stones and have used it since 1981 with good results. The procedure is safe and simple. A renal pelvic stone in a seventy-one-year-old woman was successfully destroyed by microexplosion using a percutaneous approach. We believe this is the first clinical report on the use of microexplosion for upper urinary tract calculi.

Survival time and prevention of side effects of intraperitoneal hyperthermic perfusion with mitomycin C combined with surgery for patients with advanced gastric cancer.

In an attempt to prevent postoperative intraperitoneal recurrence in patients with advanced gastric cancer and consequently to improve survival time, we treated patients with intraperitoneal hyperthermic perfusion (IPHP) using mitomycin C (MMC) combined with surgery. There were 60 patients with advanced gastric cancer who were treated with IPHP (long-term study) group, and the survival of this group was compared with the outcome in 52 patients with advanced gastric cancer treated with surgery alone (control group). To avoid or diminish side effects derived from scald injury of the peritoneal surface due to IPHP, 50 mg/kg of cimetidine was given intravenously just before administration of IPHP. For prophylaxis of anastomotic leakage, duodenostomy using a Foley catheter was performed. The 60 patients who were treated with IPHP lived longer than the 52 patients in the control group (p = 0.000610). The 3 year survival rate was 45 percent for the former compared with 16 percent for the latter. The intravenous administration of cimetidine just prior to IPHP protected the peritoneoserosal surface from scald injury, even though the heated perfusate exposure was at 44.3-46.3 degrees C for 2 hours. Because the intraabdominal pressure within the duodenum and jejunum was decompressed postoperatively through catheter duodenostomy and the peritoneoserosal surface was protected from scald injury caused by IPHP, anastomotic leakage in the study group was nil. Therefore, IPHP treatment plus aggressive surgery combined with pre-IPHP cimetidine administration are indicated for patients with advanced gastric cancer. The side effects of IPHP and postoperative morbidity can thus be reduced and a favorable outcome obtained.

Recombinant M-, B- and MB-type isozymes of human phosphoglyceric acid mutase: their large-scale production and preparation of polyclonal antibodies specific to M- and B-type isozymes.

Human phosphoglyceric acid mutase is a dimer comprising M-, B- and MB-type isozymes composed from the combination of the muscle-specific (M) and non-muscle-specific (B) subunits. Human DNAs coding M and B subunits were, respectively, reconstructed at their 5' regions without changing amino acid sequences, and expressed directly in Escherichia coli under the control of the trp promoter. M- and B-type isozymes were over-produced in the bacterial cytoplasm as soluble, active forms, which have been purified and characterized. MB-type was synthesized in vitro by recombining M- and B-type. All three recombinant isozymes thus obtained showed the same properties as the naturally-occurring ones with respect to the properties tested. Polyclonal IgGs specific to the M-type, B-type and MB-type were prepared from rabbits immunized with M- and B-type, using columns bound with M- and B-type. A method for the immunoassay of MB-type which is specifically present in cardiac muscle, is now under development.

[Changes in intestinal motility after massive small bowel resection with a reversed jejunal segment].

To evaluate the functioning and effectiveness of a 20-cm reversed jejunal segment after 75-80% massive small bowel resection (MSBR), and whether migrating polarity changes or not, we continuously measured the postoperative bowel motility (using bipolar electrodes and/or contractile strain gage force transducers) in interdigestive and postprandial conscious dogs in short- (2-5 weeks) and long-term (6-10 months) after surgery. The fasting migrating myoelectric (or motor) complex (MMC) arising from the duodenum was often interrupted at the jejunum above the proximal anastomosis and did not migrate smoothly to the reversed segment or terminal ileum. In addition, brief small discordant contractions were frequent in the jejunum above the proximal anastomosis and the proximal part of the reversed segment. The duodenal MMCs predominantly propagated to the ileum through the inherent anatomic continuity of the bowel. These findings of the MMC propagation pattern are very similar in short- and in long-term after surgery. The duration of the postprandial period without duodenal MMC activity was markedly longer in short-term, but shorter in long-term (both were significantly longer than in controls). Marked dilatation of the jejunum and reversed jejunal segment was noted across the proximal anastomosis. These results suggest that the transit time and passage of intestinal contents can be delayed and stagnated for at least 10 months after MSBR with a 20-cm reversed jejunal segment. Although, reports on the polarity of peristalsis in the reversed segment in long-term followup have been contradictory in both experimental and clinical studies, this results support the conclusion that the reversed jejunal segment maintains its inherent propagative polarity and pattern over a long postoperative period.

Successful treatment of aortoesophageal fistula resulting from aneurysm of the aortic arch.

Aortoesophageal fistulas from primary thoracic aortic aneurysms are almost always fatal. In the English literature we have found only four successfully managed cases, all descending thoracic aortic aneurysms. We report an elderly woman who developed an aortoesophageal fistula from an aortic arch aneurysm. She was successfully managed with graft replacement including reattachment of arch vessels and a staged esophagectomy which was accompanied by mobilization of the omentum into the space between the esophagus and the prosthetic graft. She was the first successfully treated patient with aortoesophageal fistula resulting from aortic arch aneurysm.

Patency after revision surgery for failing infrainguinal vein grafts.

BACKGROUND: Although the results of bypass surgery on arteries in the lower extremities have been greatly improved during the last decades, there are still a significant number of primary failures. In order to investigate the reasons for the failure of autogenous vein grafts after infrainguinal arterial bypass surgery and to ascertain the results of revision operations on the failing grafts, we conducted a clinical study. METHODS: Retrospective study on 104 patients who underwent infrainguinal arterial revascularisation with autogenous veins. RESULTS: One hundred and twenty-six bypasses were constructed in 121 limbs. Seventy-nine of these were for claudication and 47 for threatened limbs (rest pain or ischaemic tissue loss). Five-year primary and secondary patency rates were 69.7% and 84.9% respectively. Spliced or suture-repaired grafts had a poor outcome; five out of seven eventually becoming thrombosed. On the other hand, revision operations for failing but not yet thrombosed grafts produced good results; the five-year primary patency rate after these operations was 76.0%, whereas after single-segment primary grafting it was 72.1%. CONCLUSIONS: These results stress the importance of vigilant follow-up and active intervention for failing grafts. Poor outcome with injured vein grafts emphasizes the importance of meticulous vein harvesting.