Automated genomic sequence analysis of the three collagen VI genes: applications to Ullrich congenital muscular dystrophy and Bethlem myopathy.

INTRODUCTION: Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD). BM is a relatively mild dominantly inherited disorder with proximal weakness and distal joint contractures. UCMD is an autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. METHODS: We developed a method for rapid direct sequence analysis of all 107 coding exons of the COL6 genes using single condition amplification/internal primer (SCAIP) sequencing. We have sequenced all three COL6 genes from genomic DNA in 79 patients with UCMD or BM. RESULTS: We found putative mutations in one of the COL6 genes in 62% of patients. This more than doubles the number of identified COL6 mutations. Most of these changes are consistent with straightforward autosomal dominant or recessive inheritance. However, some patients showed changes in more than one of the COL6 genes, and our results suggest that some UCMD patients may have dominantly acting mutations rather than recessive disease. DISCUSSION: Our findings may explain some or all of the cases of UCMD that are unlinked to the COL6 loci under a recessive model. The large number of single nucleotide polymorphisms which we generated in the course of this work may be of importance in determining the major phenotypic variability seen in this group of disorders.

Improved molecular diagnosis of dystrophinopathies in an unselected clinical cohort.

Mutations in the DMD gene result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Readily available clinical tests detect only deletions of one exon or greater, which are found in approximately 60% of cases. Mutational analysis of other types of DMD mutations, such as premature stop codons and small frameshifting insertions or deletions, has historically been hampered by the large size of the gene. We have recently reported a method that allows the rapid and economical sequencing of the entire coding region of the DMD gene, and that is more sensitive than methods based on single-strand conformational polymorphism (SSCP) screening or other preliminary screening steps. Here we use single condition amplification/internal primer (SCAIP) sequencing analysis, in combination with multiplex amplifiable probe hybridization (MAPH) analysis of duplications, to report the frequency of mutations in a large cohort of unselected dystrophinopathy patients from a single clinic. Our results indicate that 7% of dystrophinopathy patients do not have coding region mutations, suggesting that intronic mutations are not uncommon. The availability of rapid and thorough mutation analysis from peripheral blood samples, along with an improved estimate of the percentage of non-coding region mutations, will be of benefit for improved genetic counseling and in identification of cohorts for clinical trials.

Sustained phosphorylation of mitogen-activated protein kinase is required for basic fibroblast growth factor-mediated axonal branch formation in cultured rat hippocampal neurons.

Basic fibroblast growth factor (bFGF) has been reported to promote the formation of axonal branches in cultured brain neurons. In the present study, we investigated whether the mitogen-activated protein kinase (MAPK) cascade was involved in this action of bFGF in cultured rat hippocampal neurons. Recombinant human bFGF (0.1-10 ng/ml) induced phosphorylation of p44/42 MAPK in a concentration and time-dependent manner. The phosphorylation of p44/42 MAPK occurred rapidly within 5 min after addition of bFGF, and lasted for 48 h. The bFGF-induced phosphorylation of p44/42 MAPK and axonal branch formation were both blocked by simultaneous addition of U0126 and PD98059, specific inhibitors of MAPK kinases. Furthermore, when U0126 and PD98059 were added 24 h after bFGF, phosphorylation of p44/42 m MAPK was decreased, and axonal branch formation was stopped. These results suggest that sustained activation of the MAPK cascade is required for bFGF-mediated axonal branch formation.

Spatial working memory is independent of hippocampal CA1 long-term potentiation in rats.

This study investigated the relationship between spatial working memory and hippocampal long-term potentiation (LTP) using the allocentric place discrimination task (APDT) in rats, in which the selection accuracy is a good index for spatial working memory. Either the selective M1 muscarinic receptor antagonist pirenzepine (50 microg) or the choline uptake inhibitor hemicholinium-3 (5 microg) impaired APDT selection accuracy, but neither affected the induction of LTP in the hippocampal CA1 region in anesthetized rats. In contrast, the selective N-methyl-D-aspartate receptor antagonist D-amino-5-phosphonopentanoate (200 nmol) did not impair APDT selection accuracy but completely blocked hippocampal CA1 LTP. These results suggest that spatial working memory is independent of hippocampal CA1 LTP and that the central cholinergic system is involved in spatial working memory, but not through the modulation of hippocampal CAI LTP.

Low-dose cytarabine and aclarubicin in combination with granulocyte colony-stimulating factor (CAG regimen) for previously treated patients with relapsed or primary resistant acute myelogenous leukemia (AML) and previously untreated elderly patients with AML, secondary AML, and refractory anemia with excess blasts in transformation.

We used the CAG regimen (low-dose cytarabine [10 mg/m2 per 12 hours, days 1-14], aclarubicin [14 mg/m2 per day, days 1-4], and granulocyte colony-stimulating factor [200 micrograms/m2 per day, days 1-14]) for the treatment of patients with primary resistant acute myelogenous leukemia (AML) and previously untreated elderly patients with AML, secondary AML, and refractory anemia with excess blasts in transformation (RAEB-T) in addition to relapsed AML. Forty-three of 69 (62%) patients achieved complete remission (CR), including 29 of 35 (83%) patients with relapsed AML, 1 of 8 patients with primary resistant AML, 5 of 8 elderly patients with previously untreated AML, and 8 of 18 patients with previously untreated secondary AML or RAEB-T. Ten of 22 (45%) patients > or = 65 years old achieved CR. The patients who achieved CR received at least 1 course of modified CAG therapy as the first consolidation therapy, followed by various second consolidation and intensification therapies. The median disease-free survival and overall survival were 8 and 15 months, respectively, for relapsed AML; 11 and 8 months for the elderly patients; and 8 and 17 months for secondary AML and RAEB-T. Myelosuppression was mild to moderate, and other than fever, severe nonhematologic toxicity was rare. CAG as the induction therapy seems promising for the treatment of various categories of poor-prognosis AML.

Isolation of Pythium Species from Zoysia Grass and Their Effect on Severity of Large Patch Disease.

Pythium periplocum, P. rostratum, P. torulosum, and P. vanterpoolii were predominant Pythium species isolated from nine sites with a history of large patch disease of zoysia grass. Rhizoctonia solani AG2-2 LP and the Pythium species were isolated from 21 sod samples of zoysia grass exhibiting large patch symptoms in five golf courses. R. solani AG2-2 LP was obtained from all samples, while P. periplocum, P. rostratum, P. torulosum, and P. vanterpoolii were obtained from 14, 6, 11, and 8 samples, respectively. At least one of the four Pythium species was recovered from 19 samples. To verify pathogenicity of these four species of Pythium on zoysia grass, they were inoculated alone and together with R. solani AG2-2 LP on zoysia grass. When individual isolates were used to inoculate zoysia grass, R. solani AG2-2 LP, P. periplocum, and P. vanterpoolii were moderately aggressive, while P. torulosum and P. rostratum caused little or no disease. Symptoms produced by R. solani AG2-2 LP included orange discoloration of the sheath, and the sheath was easily pulled from the crown. P. periplocum and P. vanterpoolii induced only sheath chlorosis, and the sheath was not easily removed from the crown. In coinoculation tests, the combination of R. solani AG2-2 LP and P. torulosum intensified disease severity on zoysia grass and induced more rapid symptom development than did R. solani AG2-2 LP alone. The combination of R. solani AG2-2 LP and P. periplocum or P. vanterpoolii resulted in sheath necrosis and bare patches, similar to large patch symptoms observed on golf courses.

Early impairment and late recovery of synaptic transmission in the rat dentate gyrus following transient forebrain ischemia in vivo.

Ischemic stroke causes various functional deficits in the brain such as memory impairment, and clinical reports have shown that the impaired brain functions may partially recover. However, there has been no experimental model suitable for studying cellular mechanisms of functional recovery following brain ischemia. Therefore, we investigated the long-term influence of transient forebrain ischemia on excitatory synaptic transmission in the rat dentate gyrus, a brain region relatively resistant to ischemia. Fifteen minutes of transient forebrain ischemia produced no apparent histological damage in dentate granule cells, but caused a significant reduction of basal synaptic potentials evoked by perforant path stimulation. Field excitatory postsynaptic potential remained reduced for at least 1 month after ischemia, while population spike recovered to control level in 1 month. The induction of long-term potentiation was also impaired after ischemia, but it showed faster recovery than basal synaptic potentials. In conclusion, we found that synaptic transmission in the dentate gyrus of the rat is impaired following transient forebrain ischemia, but has a potential to recover. These results may provide a good model for studying the mechanisms of impairment and recovery of brain function after transient ischemia.

Reconstruction for palmar skin defects of the digits and hand using plantar dermal grafting.

The plantar skin is considered suitable for skin grafting onto the volar aspect of the digits and hand. However, this method is not widely used because it is associated with problems at the donor site. To solve these problems, a new method was developed in which two different layers of the plantar skin are harvested from the same site. In this method, a split-thickness skin graft of the upper layer including the corneal layer of epidermis and a dermal graft of the lower layer are harvested from the same plantar skin. The split-thickness skin graft is returned to the original donor site, whereas the dermal graft is used for the palmar skin defects on the digits and hand. To prevent drying, the dermal graft was covered with a wound-covering material to achieve good graft takes. Reconstruction was performed for 17 patients using this method, involving digit-only reconstruction in 8 patients, and wider reconstruction in the other 9. Excellent color and texture match of the graft and donor sites were obtained with no noticeable marginal scarring, and the durability of the skin was satisfactory. This method was useful for skin grafting to the digits and palms with minimal sacrifice to the donor site.

Preferential inhibitory effect of soluble factor(s) in human bone marrow stromal cells on proliferation of K562 leukemia cells versus normal myeloid progenitor cells.

We examined the effect of diffusible factors generated during the culture of the KM102 stromal cell line as well as in long-term bone marrow culture (LTBMC) on K562 leukemia cells, with respect to proliferation of clonogenic cells as well as total cells, and compared it with the effect on normal myeloid progenitors (CFU-GM). Proliferation of K562 cells plated in diffusion chambers was inhibited by coculture for 3-5 days in the fluid phase of stromal cell cultures or stromal cell-conditioned medium (CM), while CFU-GM proliferation was not inhibited under the same culture conditions. The inhibitory action was not attributed to the exhaustion of nutrients or growth promoting factors such as stem cell factor. These findings suggest that bone marrow stromal cells secrete diffusible molecule(s) which exert a preferential inhibitory effect on K562 leukemic cells vs. normal CFU-GM. Neutralization with antibodies against hematopoiesis-inhibiting cytokines such as TGF-beta 1, IFN-gamma, MIP-1 alpha and IL-4 which were detected in stromal cell-CM, failed to abrogate the inhibitory effect of KM102-CM on K562 cells. IL-1, TNF-alpha, IFN-alpha and lipopolysaccharides, known as stimulators of various cytokines from stromal cells, could not enhance the inhibitory activity. Further characterization of the factors may have implications for the treatment of leukemias.

Differential effects of microtubule inhibitors on axonal branching and elongation of cultured rat hippocampal neurons.

We investigated the effects of microtubule inhibitors, taxol and colchicine, on the axonal branching and elongation in cultured rat hippocampal neurons. Taxol (50 nM) did not affect the morphology of neurons cultured under the control conditions, but significantly reduced the axonal branching stimulated by basic fibroblast growth factor. The axonal elongation stimulated by astrocyte-conditioned medium was not affected by the same concentration of taxol. Colchicine (10 nM) showed similar effects as taxol. These results suggest that microtubules play more important roles in axonal branching than in axonal elongation.

[Comparison of low-dose cytosine arabinoside and aclarubicin in combination with granulocyte colony-stimulating factor to intermediate-dose cytosine arabinoside and mitoxantrone with or without etoposide in the treatment of relapsed acute myeloid leukemia].

We used a new chemotherapy regimen for the treatment of 18 consecutive patients with relapsed AML. The regimen consisted of low-dose cytosine arabinoside (Ara-C), low-dose aclarubicin and concurrent use of G-CSF (CAG regimen). Fifteen out of 18 patients (83%) achieved complete remission (CR). Median CR duration and median survival were 6 months and 15 months, respectively. These results were similar to those of previously reported salvage therapies for relapsed AML including intensive chemotherapy consisting of intermediate-dose Ara-C and sequential mitoxantrone with or without etoposide (MC/MEC), which we previously adopted. Myelosuppression and non-hematological toxicities were apparently lower and less frequent compared to MC/MEC. The CAG regimen seems promising for the treatment of relapsed AML with its low toxicity contributing to a high quality of life for the patient.

Concurrent use of granulocyte colony-stimulating factor with low-dose cytosine arabinoside and aclarubicin for previously treated acute myelogenous leukemia: a pilot study.

We used a new chemotherapy regimen for the treatment of 18 consecutive patients with relapsed AML (median age 44 years, range 18-74). The regimen consisted of low-dose cytosine arabinoside (10 mg/m2/12 h, usually day 1 to 14), low-dose aclarubicin (10-14 mg/m2/day, day 1 to 4), and concurrent use of G-CSF (200 micrograms/m2/day) (CAG regimen). Overall, 15/18 patients (83%) achieved complete remission (CR) after one or two courses, including eight out of ten refractory patients with early relapse, second or subsequent relapses, and/or resistant relapse. Two of three patients who relapsed, achieved CR again after reinduction with a modified CAG regimen. Fourteen of the 15 complete remitters received consolidation therapy with the CAG regimen modified, followed by oral busulfan in eight cases, and by allogeneic bone marrow transplantation in two cases. At a median follow-up of 12 months, median CR duration and survival were 6 months and 17 months, respectively. Myelosuppression in the first course of induction therapy was moderate to severe. However, severe non-hematologic toxicity (WHO grade > or = 3) was characteristically rare. Although this is a preliminary study, the CAG combination seems promising for the treatment of relapsed AML, with its low toxicity contributing to a higher quality of life for the patient.

Characterization of basic fibroblast growth factor-mediated acceleration of axonal branching in cultured rat hippocampal neurons.

We analyzed in more detail the effect of basic fibroblast growth factor (bFGF) on morphogenesis of rat hippocampal neurons in dissociated cell culture. As a result, we found that bFGF selectively promoted the bifurcation and growth of axonal branches without affecting the elongation rate of primary axons. The dendritic outgrowth was rather inhibited by bFGF. These effects of bFGF resulted in increased complexity of axonal trees. The effect of bFGF was concentration dependent (0.1-10 ng/ml) and was abolished by the presence of anti-bFGF neutralizing antibody. The accelerated axonal branch formation in the presence of bFGF was restored to the basal rate following removal of bFGF, suggesting that the action of bFGF is reversible and that the continuous presence is required for bFGF to accelerate the branch formation. bFGF probably works as a progression signal rather than as a triggering signal. The bFGF-mediated acceleration of axonal branch formation was blocked by treatment with heparitinase and by tyrosine inhibitors, herbimycin A and lavendustin A, indicating the importance of heparan sulfate and tyrosine kinase in bFGF signal transduction. Treatment with a protein kinase C activator phorbol-12-myristate-13-acetate did not significantly affect the neurite branching, and the action of bFGF was not blocked by a protein kinase C inhibitor staurosporine. Protein kinase C is unlikely to play a role in branch formation. The novel action of bFGF as a regulator of axonal branching must be a particularly useful model for the study of neuritogenesis and synaptogenesis of brain neurons.

[Tuberculin sensitivity to purified protein derivatives from Mycobacterium other than tuberculosis (PPD-B, PPD-Y, PPD-F and PPD-C) and PPDs among patients with mycobacteriosis--cooperative study of the Research Committee for the Mycobacteriosis in Japan].

Purified protein derivatives (PPDs) prepared from M. intracellulare (PPD-B), M. kansasii (PPD-Y), M. fortuitum (PPD-F), M. chelonei subsp. abscessus (PPD-C) and M. tuberculosis (PPDs) were simultaneously used in skin tests on patients diagnosed as having tuberculosis or atypical mycobacteriosis to reveal their specificity, clinical usefulness and immunological status of the patients. The mean diameter of reaction (redness) for patients with M. tuberculosis positive sputum (TB group, n = 71; age, 20-90 yrs) was PPDs, 20.4 mm; PPD-B, 7.9 mm; PPD-Y, 11.7 mm; PPD-F, 0.8 mm; and PPD-C, 0.3 mm. For M. avium complex positive patients (MAC group, n = 100; age, 31-89 yrs), the results were PPDs, 10.9; PPD-B, 16.9 mm; PPD-Y, 10.7 mm; PPD-F, 1.6 mm; and PPD-C, 0.3 mm. The M. kansasii positive patients (K group; n = 8) showed results of PPDs, 12.6 mm; PPD-B, 10.7 mm; PPD-Y, 20.8 mm; PPD-F, 0.5 mm; PPD-C, 0.0 mm. The M. fortuitum positive patients (F group; n = 5) had measurements of PPDs, 5.8 mm; PPD-B, 4.4 mm; PPD-Y, 9.8 mm; PPD-F, 17.8 mm; and PPD-C, 16.0 mm. The patients who were previously M. tbc. positive but presently negative patients (pre. TB group; n = 50) showed the following results: PPDs, 16.6 mm; PPD-B, 7.4 mm; and PPD-Y, 10.9 mm. For the patients who were previously M. avium complex positive (previous MAC group; n = 19), the results were PPDs, 10.4 mm; PPD-B, 9.9 mm; and PPD-Y, 7.7 mm. Also considering their frequency distribution curve, with exception of the previous MAC group, the patient groups showed specificity to the PPD of the bacilli detected. The previous MAC group recorded no significant difference in response to PPDs and PPD-B. Strong cross reactions were observed between PPD-F and PPD-C, and moderate reactions between PPDs, PPD-B and PPD-Y. Cross reactions were scarce between PPDs, PPD-B or PPD-Y and PPD-F or PPD-C. Though it is difficult to distinguish cross-reaction and multiple infections, majority of the patients (72-85%) showed greatest response to the PPD that corresponds with the species of bacilli detected. In conclusion, two or more PPDs applied simultaneously can be of aid in diagnosing mycobacteriosis especially in the early stages of the disease. Also, cross-reactions between atypical mycobacteria and PPDs should be taken into consideration when diagnosing infection caused by M. tuberculosis.

[Therapeutic results of DCMP therapy (2M-80) in acute non-lymphocytic leukemia--the relationship between the residual leukemic cells and duration of remission].

In this study the duration of complete remission by daunomycin, cytosine arabinoside (Ara-C), 6MP and prednisolone (DCMP) therapy using a large dose of Ara-C (200 mg) (protocol 2M-80) was compared with that of DCMP therapy with a low dose of Ara-C (80-160 mg) in acute non-lymphocytic leukemia (ANLL). In protocol 2M-80, the chemotherapy was continued until leukemic blasts in marrow were attained below 3%. Complete remission was induced in about 80% of ANLL patients in both chemotherapies. However, the duration of remission in protocol 2M-80 appeared to be much longer than that of DCMP therapy with a low, dose of Ara-C. This difference was dependent not on the consolidation and maintenance therapy, but on the total dose of Ara-C used and leukemic blasts left in marrow at the end of chemotherapy. This suggests that it is important to reduce leukemic blasts in marrow as low as possible by induction chemotherapy to obtain a long-term remission in ANLL.