Molecular cloning and expression of a novel hydroxymethylcytosine-specific restriction enzyme (PvuRts1I) modulated by glucosylation of DNA.

The kanamycin resistance plasmid Rts1 restricts the growth of bacteriophage T2, T4 and T6. The DNA of these phage contains hydroxymethylcytosine (HMC) in place of regular cytosine and is modified by glucosylation. When HMC is not glucosylated, as in the DNA of glucosyl transferase-deficient T4 phage, this restriction becomes less apparent, a phenomenon not observed with any other known restriction systems. On the other hand, glucosylation of HMC in T6 phage leads to a less efficient restriction, while restriction of bacteriophage T2 remains unchanged. The modulating effect of glucose cannot be seen when cells contain a large amount of this enzyme, as in the case when multiple copies of its determinant are present in the cells. T-odd phage and bacteriophage lambda are not restricted by Rts1 suggesting that the restriction is specific to DNA containing HMC. The restriction phenotype is due to a single gene coding for a polypeptide of 293 amino acids. This enzyme has been named PvuRts1I. A gene with the sequence motifs similar to modification enzymes was found upstream of the gene coding for PvuRts1I. This gene, however, neither modifies the restriction phenotype of PvuRts1I, nor codes for detectable modification enzyme. T4 mutants with increased resistance to PvuRts1I appear to have deficiency in their beta-glucosyl transferase enzyme.

Trigger digits-associated carpal tunnel syndrome: relationship between carpal tunnel release and trigger digits.

Of 875 idiopathic carpal tunnel syndrome (CTS) cases, 101 (11.5%) required trigger digit release operations within three years before and/or after carpal tunnel release (CTR); these 101 cases were investigated, retrospectively. Trigger digit release (TDR) was performed most often after the CTR, especially within three months. Next most common was at the same time as the CTR. The TDR performance rate after CTR was 5.9%. The nerve conduction study (NCS) comparison between trigger digits-associated CTS and isolated CTS showed that pre-operative distal motor latency was significantly more delayed in trigger digits-associated CTS, while there was no evidence of any difference due to age or gender. The difference of operative method (open or endoscopic procedure) did not influence the incidence rate of trigger digits after the CTR. This study suggested that trigger digits-associated CTS has a previously developed wide-ranging narrowing of the flexor tendon sheath.

A new precipitation method with magnetic separation for high-density-lipoprotein cholesterol assay.

We describe a new precipitation method for high-density-lipoprotein cholesterol quantitation. The new method uses magnetic force instead of centrifugal force to separate high-density-lipoprotein from other lipoproteins that are fractioned with a precipitating reagent. The reagents used for the new method are the same as those for the conventional method except that magnetizable particles are included in the former. The magnetizable particles are used without any modifications. The correlation between the new and the centrifuge methods with dextran sulfate-magnesium chloride, sodium phosphotungstate-magnesium chloride and polyethylene glycol 6,000 were satisfactory (r = 0.990, 0.997 and 0.997, respectively). The new method, which can be combined with any precipitating reagents used in conventional methods, is very simple to perform and does not need any special equipment.

Amyloidosis complicating idiopathic myelofibrosis.

Three cases of amyloidosis occurring in the later course of idiopathic myelofibrosis were studied at autopsy. In these cases, the amyloid deposition was seen only in the renal glomerulus, which had caused proteinuria. Although their exact nature could not be determined by immunohistochemistry, the amyloid deposits in the three cases were permanganate resistant and exhibited the same organ distribution, indicating a similarity in their character. These cases suggest that amyloidosis might be a complication of idiopathic myelofibrosis, implying that idiopathic myelofibrosis could be a disorder underlying amyloidosis.

Does electrical stimulation of the sciatic nerve prevent suspension-induced changes in rat hindlimb bones?

Osteoporosis due to mineral loss is a major health problem resulting from long-term spaceflight. The development of a suitable countermeasure is essential because an advanced decrease in bone density could be irreversible. Therefore the current study was performed to test our hypothesis that the loading of bones by electrical stimulation-induced muscle contraction may prevent the mineral loss caused by gravitational unloading and bone growth will be maintained. During 10 d of hindlimb suspension, electrical stimulation at 1, 50, or 100 Hz was administered through the left sciatic nerve at the gluteal region of rats with approximately 300 g body weight. The dry weight, mineral content, and mineral density in hindlimb bones were analyzed. The dry weight and mineral content of femur and tibia-fibula in hindlimb-suspended rats tended to be less than in the age-matched cage controls. However, these detrimental effects were prevented by stimulation at 50 and 100 Hz. A positive effect of stimulation was seen even in the nonstimulated limb, although greater effect was induced in the stimulated limb. It is suggested that loading by stimulation-induced muscle contraction at higher frequencies is beneficial for the maintenance of bone growth or the prevention of mineral loss, or both, during hindlimb suspension in rats.

[Clinical evaluation of a reagent for detection of DNA of Mycobacterium tuberculosis complex using the ligase chain reaction (LCR) method].

We evaluated the clinical efficacy of LCR MTB, a reagent developed by Abbott in the USA, in the full automatic ligase chain reaction (LCR) for detection of DNA of M. tuberculosis complex using a thermostable ligase. Using 458 samples isolated from patients with tuberculosis, LCR was compared with a smear method and with a culture method, and was also compared with two other methods of gene amplification, MTD and Amplicor, using 340 and 200 of the 458 samples, respectively. The LCR method detected M. tuberculosis in 49.8% (228/458) of the samples, and was superior to the smear method (31.9%, 146/458) and the culture method (39.1%, 179/458) in sensitivity. The LCR method was also superior to the MTD and Amplicor methods; sensitivity were 37.9% (129/340) for MTD vs. 47.6% (162/340) for LCR, and 56.5% (113/200) for Amplicor vs. 59.5% (119/200) for LCR. These favorable results and the convenience of the LCR method, which enables rapid detection of target genes with a high degree of sensitivity, strongly suggest that LCR MTB is useful as a reagent for detection of M. tuberculosis using nucleic acid amplification.

[Investigation of bone mineral distribution in Japanese using dual-energy X-ray absorptiometry].

To investigate bone mineral distribution in humans, the authors conducted a cross-sectional survey of, and performed bone-density measurements on, 1,310 healthy Japanese ranging in age 5 to 85 years. Eight hundred fifty-eight of the subjects were female, and 452 were male. Arm, leg, and spine bone mineral content (BMC) and bone mineral density (BMD) were assessed by dual-energy X-ray absorptiometry (DXA), and the subjects were divided into 5-year age groups. BMD showed increases with skeletal growth until reaching a peak at 15 to 19 years in females, and 25 to 29 for males. For both sexes the fastest growth to maturity in terms of bone mass values was in the late 20s. Females, though, had higher arm, leg, and spine remodeling rates than males. In premenopausal women no changes in arm, leg or spine BMC and BMD were observed. Postmenopausal women showed an overall reduction in bone mass, most noticeably in the spine. After menopause, women had about 10 years of accelerated loss (1.46%/year). Vertebral BMD values were similar for men and women (1.10 +/- 0.20g/cm2 for males vs. 1.09 +/- 0.14g/cm2 for females, p > 0.05). BMC values were significantly higher in males, and males at all times had a higher arm and leg BMD. There were no significant value differences in either sex for left and right leg BMC and BMD; however, from the age of 15, right arm values were significantly higher likely due to right handedness. For both sexes the order of BMC and BMD was leg, spine, and arm.

[A Japanese language version of the health-promoting lifestyle profile].

The development and initial psychometric evaluation of a Japanese version of the Health-Promoting Lifestyle Profile II (HPLP II) is described. The 52-item instrument was translated into Japanese and was found to be culturally relevant and reliable in a pilot study. The Japanese version was then administered to adiverse but predominantly Japanese group of 337 subjects residing in northern Japan. The Japanese version of the HPLP II was evaluated using factor analysis and reliability measurement. Six factors similar to those isolated previously during psychometric assessment of the English language version were extracted. Those six dimensions comprise the HPLP II subscales of: 1. Health responsibility, 2. Spiritual growth, 3. Physical activity, 4. Interpersonal relations, 5. Nutrition, and 6. Stress management. The alpha reliability coefficient for the total scale was 0.94 and the 2-week retest reliability was 0.91; the alpha coefficients for the subscales ranged from 0.70 to 0.87. The Japanese language version of the HPLP II appears to have sufficient validity and reliability for use by researchers who wish to describe the health-promoting components of lifestyle among the Japanese population and to explore differences and similarities in the health-promoting lifestyle of Japanese and American subjects or those of other ethnic groups. Further evaluations of measurement with different populations appears warranted. This instrument will enable researchers to investigate patterns and determinants of health-promoting lifestyle, as well as the effects of interventions to alter the lifestyle.

[Pathophysiology and diagnosis of malignant neoplastic disorders of the hematopoietic system].

The Pathophysiological analysis of hematopoietic tumors has advanced markedly owing to the progress in the fields of histomorphology, biochemistry, hematopoietic stem cells including cytokines, cell surface markers, cytogenetics, and molecular biology. This progress has led to the establishment of many new disease concepts and has almost completely changed the recognition of hematopoietic tumors. These basic studies have also supported the development of effective therapy of hematopoietic tumors. Of course, this progress has been reflected in the techniques and knowledge in routine laboratory evaluation.

[Evaluation of a one step sandwich enzymeimmunoassay for serum erythropoietin--serum erythropoietin values in polycythemia vera and related hematological disorders].

We evaluated a newly developed enzymeimmunoassay for serum erythropoietin (Epo) and investigated relationship between Epo levels and hematological disorders. This method has several advantages including simplicity, high sensitivity, good precision. Moreover, the procedure requires only about 2.5 hours. Samples from 134 healthy subjects showed a normal logarithmic distribution, and its normal range was 4.5 approximately 21.3 mU/ml. The levels of Epo in normal subjects and various hematological disorders were as follows: 10.5 +/- 4.1 (mean +/- SD mU/ml) in normal subjects, 2.2 +/- 1.7 in polycythemia vera (PV), 6.1 +/- 3.1 in essential thrombocythemia, 17.8 +/- 27.3 in chronic myelogeneous leukemia, 3.6 +/- 1.8 in stress erythrocytosis, 39.4 and 14.1 in two cases of primary myelofibrosis, 1289 +/- 4798 in iron deficiency anemia and 6564 +/- 10870 in aplastic anemia. In patients with PV, serum Epo were low and did not correlate with hemoglobin concentration. However, inverse correlation was found between changes of Epo levels and hemoglobin levels in most patients. In cases in which PV progressed into myelofibrosis, anemia developed and Epo levels increased accordingly. These results suggest that the method is thought to be useful and reliable for the diagnosis and monitoring of PV and related hematological disorders.

[Clinical significance of red cell distribution width in polycythemia vera].

We evaluated changes in red cell distribution width-standard deviation (RDW-SD) measured using a multiple parameter automated hematology analyzer E 4000 in patients with polycythemia vera (PV). Patients with iron deficiency anemia, those with chronic myelogenous leukemia, those with primary thrombocythemia, and normal subjects were examined as controls. In the patients with PV, as in those with the other 3 diseases, RDW-SD tended to be higher than in the normal controls when red blood cell counts were high. The RDW-SD in patients with PV transiently increased following administration of a myelosuppressive, which corresponded to the transition period from microcytes to normal blood cells. It was even higher during the polycythemic period than during the myelofibrotic period. This may be associated with hematopoietic abnormality due to extramedullary hematopoiesis. RDW-SD seems to well reflect the pathologic status of PV.

[Assessment of laboratory findings in megaloblastic anemia--measurement of serum vitamin B12 and methylmalonic acid].

The laboratory findings of 20 patients with untreated megaloblastic anemia due to vitamin B12 deficiency were analysed. The material consists of 13 patients with pernicious anemia, 6 with postgastrectomy B12 deficiency and one with malabsorption syndrome. Hematological data (RBC, Hgb, Ht, WBC, Plt) were correlated with each other and serum LDH levels. Megaloblastic changes of bone marrow were apparent in cases of which Hgb values were below 9 g/dl, although its change were not clear in cases with mild anemia (above 9 g/dl). However, giant metamyelocytic changes of bone marrow were seen even in cases with mild anemia. Serum B12 levels in 6 out of 19 cases (31.6%) measured by clinical laboratory center were within normal range. In contrast, its level in all cases measured by radiodilution assay using R-protein or intrinsic factor were lower than normal values. Serum B12 levels measured by the latter method were correlated with various hematological data and also related with hematological severity, although its level measured by clinical laboratory did not have any correlation with hematological data. Schilling test seemed to be unreliable, because sample volume which was suggested by kit manual was too small (2 ml) to catch enough radioactivity for accurate measurement. Serum methylmalonic acid levels measured by gas capillary mass spectrophotometry were higher than normal values in all cases and were well correlated with hematological data.

[Molecular evidence for a single clonal origin in a patient with multiple myeloma and non-Hodgkin's lymphoma].

A 49-year old man was admitted in November 1989, because of anemia, abnormal shadowing on chest X ray and hyperproteinemia. Biclonal gammopathy (IgG kappa + IgA kappa) was shown in serum, and Bence Jones protein in urine. The bone marrow examination showed an increased number of abnormal plasma cells (15.7%) and no evidence of lymphoma, A diagnosis of multiple myeloma (MM) was made. In April 1990, while the patient was treated with the modified M2 regiman, swelling of the right cervical lymph node was observed. Lymph node biopsy revealed that he had non-Hodgkin's Lymphoma (:NHL, diffuse, mixed, B cell type). He was retreated with the CHOP regimen for both disease, but died of respiratory failure in October. 1991. To establish the clonal origin of this case of concominant MM and B-cell NHL, the immunoglobulin gene rearrangements in his lymph node and bone marrow were analyzed. Southern blot analysis with the JH probe and Ck probe showed one common band and one different band in the two samples. Our data suggest that two B-cell malignancies may have arisen from a single B-cell progenitor.