Complications of laser conization versus loop electrosurgical excision procedure in pre- and postmenopausal patients.

Purpose ofinvestigation: The aim of this retrospective study was to compare the results and complications of laser conization and loop electrosurgical excision procedure (LEEP), performed for cervical intraepithelial neoplasia (CIN) or microinvasive carcinoma, between postmenopausal and premenopausal patients. MATERIAL AND METHODS: This study recruited a total of 551 patients. In the laser group (n = 405), there were 361 (89.1%) premenopausal and 44 (10.9%) postmenopausal women. In the LEEP group (n = 146), there were 129 (88.4%) premenopausal and 17 (11.6%) postmenopausal women. The factors investigated in both groups were the length of the tissue cone removed and the presence of positive endocervical cone margins, residual disease, and cervical stenosis. RESULTS: In the laser group, the length of the tissue cone was significantly longer in postmenopausal patients (17.9 ±3.9 mm vs. 15.7 ± 3.6mm; p = 0.002). The rate of positive endocervical margins was significantly higher in premenopausal patients (9.1% vs. 0%; p = 0.037). The rate of cervical stenosis was significantly higher in postmenopausal patients (59.1% vs. 8.3%; p < 0.0001). In the LEEP group, there were no differences in the length of the tissue cone (premenopausal, 11.7 ± 1.9 mm vs. postmenopausal, 11.4 ± 2.7 mm; p = 0.12), the rate of positive endocervical margins (24.0% vs. 17.6%), or the rate of residual disease (13.2% vs. 17.6%). The rate of cervical stenosis was significantly higher in postmenopausal patients (23.5% vs. 4.1%; p = 0.002); however this rate was significantly lower than that seen in the laser group. CONCLUSION: In postmenopausal patients, the rates of positive endocervical cone margins and of residual disease were higher in the LEEP group; however, the rate of cervical stenosis was higher in the laser group. Physicians should be aware of the characteristics of the devices used for cervical conization in postmenopausal women with CIN.

Preliminary study for the assessment of physical activity using a triaxial accelerometer with a gyro sensor on the upper limbs of subjects with paraplegia driving a wheelchair on a treadmill.

OBJECTIVE: This study aimed to examine whether, on the basis of the relationship between sensors attached on the upper limbs and energy expenditure (EE) at the time of wheelchair propulsion, there are differences in the measurement of EE depending on the sensor attachment site and whether addition of the angular velocity information to the acceleration value is advantageous. We also aimed to clarify the variables used to estimate EE as well as the estimated error. SETTING: Laboratory of the National Hospital Organization Murayama Medical Center, Japan. METHODS: Six male subjects with spinal cord injuries participated in the study. Each wore sensors at the wrist and the middle upper arm on both sides while driving a wheelchair on a treadmill at three levels: very, very light; very light; and fairly light. Triaxial acceleration, triaxial angular velocity and EE were measured during driving. We analyzed the correlation between EE and acceleration, angular velocity and synthesized values of acceleration and angular velocity at each location using regression, multiple regression and Bland-Altman analyses. RESULTS: The determination coefficients between EE and the acceleration, angular velocity and synthesized values of acceleration and angular velocity varied from 0.68 to 0.87 at each location. The mean difference between the measured and estimated EE varied from 0.0028 (s.d., 0.0027) kcal min(-1) kg(-1) on the right upper arm. CONCLUSION: These findings suggest that combining the synthesized values of angular velocity and acceleration of the motion sensors on the upper limbs might reflect EE during a wheelchair driving activity on a treadmill.

Production method of millimeter-wave absorber with 3D-printed mold.

We established a production method of a millimeter-wave absorber by using a 3D-printed mold. The mold has a periodic pyramid shape, and an absorptive material is filled into the mold. This shape reduces the surface reflection. The 3D-printed mold is made from a transparent material in the millimeter-wave range. Therefore, unmolding is not necessary. A significant benefit of this production method is easy prototyping with various shapes and various absorptive materials. We produced a test model and used a two-component epoxy encapsulant as the absorptive material. The test model achieved a low reflectance: ∼1% at 100 GHz. The absorber is sometimes maintained at a low temperature condition for cases in which superconducting detectors are used. Therefore, cryogenic performance is required in terms of a mechanical strength for the thermal cycles, an adhesive strength, and a sufficient thermal conductivity. We confirmed the test-model strength by immersing the model into a liquid-nitrogen bath.

Tissue-specific and high-level expression of the human tyrosine hydroxylase gene in transgenic mice.

Transgenic mice carrying multiple copies of the human tyrosine hydroxylase (TH) gene have been produced. The transgenes were transcribed correctly and expressed specifically in brain and adrenal gland. The level of human TH mRNA in brain was about 50-fold higher than that of endogenous mouse TH mRNA. In situ hybridization demonstrated an enormous region-specific expression of the transgene in substantia nigra and ventral tegmental area. TH immunoreactivity in these regions, though not comparable to the increment of the mRNA, was definitely increased in transgenic mice. This observation was also supported by Western blot analysis and TH activity measurements. However, catecholamine levels in transgenics were not significantly different from those in nontransgenics. These results suggest unknown regulatory mechanisms for human TH gene expression and for the catecholamine levels in transgenic mice.

Myocardial beta-adrenergic receptor function during the development of pacing-induced heart failure.

The development of pacing-induced heart failure was studied in chronically instrumented, conscious dogs paced at a rate of 240 beats/min for 1 d (n = 6), 1 wk (n = 6), and 3-4 wk (n = 7). Left ventricular (LV) dP/dt was decreased (P < 0.0125) at 1 d, LV end-diastolic pressure and heart rate were increased (P < 0.0125) at 1 wk, but clinical signs of heart failure were only observed after 3-4 wk of pacing. Plasma norepinephrine rose (P < 0.0125) after 1 d of pacing, whereas LV norepinephrine was reduced (P < 0.0125) only after 3-4 wk of pacing. Both the fraction of beta-adrenergic receptors binding agonist with high affinity and adenylyl cyclase activity decreased (P < 0.0125) after 1 d of pacing. Total beta-adrenergic receptor density was not changed at any time point, but beta 1-adrenergic receptor density was decreased (P < 0.0125) after 1 wk. The functional activity of the guanine nucleotide binding protein, Gs, was not reduced, but the Gi alpha 2 isoform of the alpha subunit of the GTP-inhibitory protein rose after 3-4 wk of pacing. Thus, myocardial beta-adrenergic signal transduction undergoes change shortly (1d) after the initiation of pacing, before heart failure develops. The mechanism of beta-adrenergic receptor dysfunction in pacing-induced heart failure is characterized initially by elevated plasma levels of catecholamines, uncoupling of beta-adrenergic receptors, and a defect in the adenylyl cyclase catalytic unit. Selective down-regulation of beta 1-adrenergic receptors, increases in Gi alpha 2, and decreases in myocardial catecholamine levels occur as later events.

Downregulation of adenylylcyclase types V and VI mRNA levels in pacing-induced heart failure in dogs.

We have shown that the heart expresses two distinct forms of adenylylcyclase mRNA, types V and VI. In this study we have characterized the expression of these two mRNA species in heart failure generated by overdrive pacing at a rate of 240 beats/min. After 4 wk, left ventricular end-diastolic pressure and heart rate increased significantly with the appearance of signs of heart failure, i.e., edema, ascites, and exercise intolerance. Basal as well as forskolin-stimulated adenylylcyclase activities decreased significantly, which was accompanied by a reduction in the steady state mRNA levels of adenylylcyclase types V and VI. These data suggest that in this model of cardiomyopathy, the downregulation of adenylylcyclase catalytic activity results, at least in part, from a reduction in the steady state levels of types V and VI adenylylcyclase mRNA levels.

Novel alternative promoters of mouse glial cell line-derived neurotrophic factor gene.

We previously isolated cDNA and genomic DNA of the mouse glial cell line-derived neurotrophic factor (GDNF) gene and found that the gene consists of three exons. Recently, it was suggested that an alternative promoter exists within intron 1 of the human GDNF gene, but this has not been confirmed. Novel cDNA clones of the mouse GDNF gene were isolated by 5'-rapid amplification of cDNA ends from postnatal day-14 striatum. A novel exon, containing 351 nucleotides, exists between exon 1 and exon 3 (referred to as exon 2 in our previous report). Luciferase reporter assay showed that a core promoter for the novel exon 2 requires its 5'-untranslated region. Primer extension analysis and reverse transcription-PCR identified another novel transcript that starts 39 bp upstream of exon 3, and the core promoter activity exists within a region containing putative Sp1 sites. Although the core promoters for the novel exons are different from those previously identified, transcripts derived from each promoter coincidentally increased with interleukin-1beta or tumor necrosis factor-alpha stimulation. Gel retardation assays suggested that the NF-kappaB binding site in intron 1 would be involved in the cytokine response of the mouse GDNF gene.

L-gulonolactone oxidase activity and vitamin C status in riboflavin-deficient rats.

The effect of riboflavin deficiency on the activity of L-gulonolactone oxidase [L-gulono-gamma-lactone: oxygen 2-oxidoreductase, EC 1.1.3.8] and on vitamin C status was studied. A marked decrease in the specific activity of L-gulonolactone oxidase was observed in the liver microsomes isolated from riboflavin-deficient rats: the specific activity was approx. one-third of that in the microsomes isolated from control rats. The L-ascorbic acid content in the liver of the riboflavin-deficient rats was approx. one-half of that in the liver of the control rats. It seems that the rate of production of L-ascorbic acid in the riboflavin-deficient rats is limited by the decreased level of L-gulonolactone oxidase activity. Immunotitration using rabbit antiserum directed to L-gulonolactone oxidase revealed that a substantial amount of an inactive form of this enzyme is present in the liver microsomes of the riboflavin-deficient rats. L-Gulonolactone oxidase activity in the microsomes of these rats increased by approx. 35% upon addition of FAD, but it was slightly decreased by the addition of FMN or riboflavin. These results indicate that the liver microsomes of the riboflavin-deficient rats contain a protein which exhibits L-gulonolactone oxidase activity upon addition of Fad.

Cloning and structural organization of the gene encoding the mouse glial cell line-derived neurotrophic factor, GDNF.

cDNA for glial cell line-derived neurotrophic factor (GDNF) was cloned from mouse neonatal brain by the method of 5'-rapid amplification of cDNA end (5'-RACE), and the sequence of it's 5'-untranslated region (5'-UTR) was determined. The mouse GDNF gene was then isolated from a genomic library and analyzed for its nucleotide sequence. In vitro translation analysis indicated that the second ATG codon in an open reading frame is the translation start point. Structural analysis of the isolated clones showed that the GDNF gene was separated into three exons and the actual translation start point was present in the second exon. RNA blot hybridization analysis indicated that the GDNF mRNA is approximately 4.5 kb long. The transcriptional start site in the GDNF gene was determined and a typical TATA box sequence was found in the promoter region. On the other hand, the gene expression of GDNF in C6 glioma cells was transiently induced by treatment with phorbol myristate acetate (PMA), but not by forskolin.

Expression of four types of human tyrosine hydroxylase in COS cells.

Alternative splicing from a single gene produces four kinds of human tyrosine hydroxylase (types 1-4), which have structural diversity only in the N-terminal region. We attempted expression of the type 1-4 enzymes in COS cells and performed kinetic analyses. All had enzymatic activities. The Km values of the four types for L-tyrosine and 6-methyl-5,6,7,8-tetrahydropteridine were similar, although their relative homospecific activities were clearly different. The type 1 enzyme displayed the highest activity.

A family with beta-galactosidase deficiency: three adults with atypical clinical patterns.

Three adult patients in a single family showed severe myoclonus, ataxia, and pyramidal signs. Enzymatic analysis of lymphocytes, plasma, and cultured skin fibroblasts showed marked deficiency of beta-galactosidase activity, more profound with GM1 ganglioside than with another natural substrate, asialofetuin. Other lysosomal hydrolases were normal. Although the physical signs were similar to those of types 1 and 2 GM1 gangliosidosis, none had bony abnormalities.

Value of cardiac ultrafast computed tomography for detecting right atrial thrombi in chronic atrial fibrillation.

We evaluated the accuracy of cardiac ultrafast computed tomography in diagnosing atrial thrombi in 70 patients with chronic atrial fibrillation, and identified the predictors of atrial thrombi from among clinical, echocardiographic, and ultrafast computed tomographic features. Ultrafast computed tomography identified 11 atrial thrombi in 9 patients: 4 patients had thrombi in the left atrium, 3 in the right, and 2 in both. Transthoracic echocardiography detected only 4 left atrial thrombi, and enlargement of the left or right atrium was associated with atrial thrombi (p <0.05).

The 5'-untranslated region of the mouse glial cell line-derived neurotrophic factor gene regulates expression at both the transcriptional and translational levels.

We previously cloned mouse glial cell line-derived neurotrophic factor (GDNF) cDNA and genomic DNA and found that the mouse gene contains a 1086-bp 5'-untranslated region (5'-UTR). We investigated the contributions of the 5'-UTR to promoter activity and found one positive regulatory region and two negative regulatory regions in the 5'-UTR. In the present study, using gel retardation assays and mutation analyses, two novel cis-elements that interact with nuclear extracts from mouse astrocytes were identified. The first cis-element (nucleotides (nt) +70 to +81) enhances promoter activity, whereas the second cis-element (nt +239 to +247) attenuates promoter activity in a position- and orientation-dependent manner. Suppression of gene expression by a third region (nt +509 to +580) occurs at the translational level. The ATG sequence (nt +547 to +549) has the potential to initiate translation and to attenuate the efficiency of translation for the GDNF precursor coding region. Furthermore, we identified an alternative promoter in the 5'-UTR that is driven by an Sp1 element, circumventing the translational suppression. Taken together, the 5'-UTR of mouse GDNF contains two novel cis-elements, a short upstream open reading frame and an alternative promoter that influences gene expression at both the transcriptional and translational levels.

Promoter analysis and characteristics of the 5'-untranslated region of the mouse glial cell line-derived neurotrophic factor gene.

We have cloned the mouse GDNF cDNA and genomic DNA to study the molecular mechanism of gene expression. Primer extension and RT-PCR analyses indicated that the mouse gene contains 1086 bp of 5'-untranslated region (5'-UTR) [Gene 203 (1997) 149]. In this report, we identified the core promoter region of mouse GDNF and examined the role of the 5'-UTR in gene expression. Promoter deletion analyses indicated that the proximal region (-81 to +28), which includes a TATA-box, is necessary for high-level expression of GDNF. Using reporter constructs encoding luciferase or fusion gene of GDNF to enhanced green fluorescent protein that were transiently transfected to mouse astroglial cell-line TGA-3 cells and rat glioma C6 cells, we investigated effects of the 5'-UTR on promoter activity. Luciferase reporter assay indicated that a region downstream of the transcription initiation site may include a positive regulatory element, while two more distal regions appear to contain negative regulatory elements, which was correlated to the mRNA level based on RNase protection assay. Both negative regulatory elements attenuated promoter activity in a position-dependent manner. Nuclear proteins from C6 glioma cells were shown to interact with several regions (+65/+105, +233/+265, and +554/+582) including each of the regulatory elements, suggesting that regulation of GDNF expression by the 5'-UTR occurred mainly at the transcriptional level.

Nitric oxide via macrophage iNOS induces apoptosis following traumatic spinal cord injury.

To investigate the pathophysiological mechanisms involved in post-traumatic impairment of the spinal cord, we analyzed expression patterns of the inducible nitric oxide synthase (iNOS) gene following acute injury of rat spinal cord using a weight drop technique. PCR analysis revealed that iNOS mRNA appeared at 3-12 h after injury and declined thereafter. Immunohistochemical analysis showed that iNOS-positive cells invaded the lesioned area through the perivascular space at 6 h after injury. The population of these cells peaked at 24 h and then declined to disappear 3 days after injury. The iNOS-positive cells were also stained with ED-2 but not with ED-1 or OX-42, indicating that these cells were macrophages and/or perivascular cells. In parallel with the appearance of iNOS-positive cells, other cells emerged that were positively stained by the terminal deoxynucleotidyl-transferase-mediated dUDP-biotin nick end-labeling (TUNEL) assay. TUNEL-positive cells were scattered in the lesioned area 1 day after injury, but some in the surrounding area close to iNOS-positive cells. Administration of L-Ng-nitro-arginine methylester, a competitive inhibitor of NOS, resulted in a reduction of TUNEL-positive cells in the lesioned area. These results suggest that nitric oxide generated by iNOS of macrophages and/or perivascular cells plays a significant role in eliminating damaged cells from the lesioned area by apoptosis.

Effectiveness of transcoronary chemoembolization for metastatic right ventricular tumor derived from hepatocellular carcinoma.

A right ventricular outflow tract was partially obstructed by a metastatic tumor from a hepatocellular carcinoma. This tumor was treated via chemoembolization of the right coronary artery, which resulted in tumor regression and improvement of the patient's symptoms.

Promotion of proliferation of murine hematopoietic stem cells by growth factors in murine amniotic fluid.

It has been shown that murine amniotic fluid (MAF) displays immunosuppressive activities. We examined the effect of MAF obtained from normal mice on a murine fetal liver cell (FLC) primary culture in vitro for the detection of the possible existence of cytokines that affect hematopoiesis. MAF promoted proliferation of murine FLC and adult bone marrow cells. Proliferation-promoting activity of MAF was observed throughout the period between days 12 and 15 of pregnancy, peaking at day 12. Pooled MAF was subjected to purification procedures to isolate the active molecule(s). After ion exchange and size exclusion chromatography, one of the active molecules was shown to react with anti-mouse stem cell growth factor (SCF) antibody and the molecular weight was determined as 40-45 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The proliferation-promoting activity of MAF was partially neutralised by the anti-SCF antibody. These results provide evidence that MAF may contain multiple growth factors for FLC, one of which may be a unique molecule related to SCF.

Ferrous ion activates the less active form of human adrenal tyrosine hydroxylase.

The less active form of human tyrosine hydroxylase has been previously reported but its physiological role is unknown. We partially purified the less active form of tyrosine hydroxylase from human adrenals, and examined differences in the properties of the active and less active forms. We succeeded in activation of the less active form of human tyrosine hydroxylase by addition of 100 ?M Fe(2+). Fe(2+) decreased the K(max) for pteridine cofactor in both the active and less active forms, but increased the V(max) only in the less active form. Fe(2+) changed the V(max) but not the K(m) of the less active form for tyrosine. These results suggest that Fe(2+) may regulate tyrosine hydroxylase activity in vivo as a result of activation of its less active form.

Intracerebrally administered (6r)-l-erythro-tetrahydrobiopterin does not affect extracellular levels of dopamine and serotonin metabolites in rat striatum in vivo during measurement by brain micro-dialysis system.

By the use of the brain micro-dialysis technique combined with HPLC, the changes in the extracellular levels of dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and a serotonin(5-HT) metabolite, 5-hydroxyindoleacetic acid (5-HIAA) were examined in the rat striatum before and after intracerebral injection of a vehicle or (6R)-l-erythro-tetrahydrobiopterin (6R-BH(4)), the natural form of the cofactor for the tryrosine hydroxylase and tryptophan hydroxylase. No apparent change after the 6R-BH, treatment was found in the levels of DA, DOPAC, HVA and 5-HIAA in the striatal dialysate. In contrast, the levels of total biopterin in both the operated (dialysis probe-implanted) and unoperated striatum of 6R-BH(4)-treated rats increased by 23- and 93-fold, respectively, when compared with those of the control, vehicle-treated rats. The results indicate that increased levels of the tetrahydrobiopterin cofactor may not affect the release of DA and the extracellular level of DA and 5-HT metabolites in the physiologically normal brain.

Sandwich enzyme immunoassay of dopamine-?-hydroxylase in cerebrospinal fluid from control and parkinsonian patients.

A sandwich enzyme immunoassay (EIA) was established by using purified human serum dopamine-?-hydroxylase (DBH) as a standard protein and a monospecific polyclonal antibody raised against human DBH purified from human pheochromocytoma. The EIA was applied to measuring DBH levels in human CSF from Parkinsonian patients and control patients devoid of neurological diseases. The control group had DBH content of 21.1 +/- 3.1 ng/ml CSF and DBH activity of 24.0 +/- 3.7 ? U/ml CSF, and Parkinsonian group 3.3 ? 0.7 ng/ml CSF (16% of control) and 4.6 +/- 0.7 ?U/ml CSF (19% of control) (mean +/- SEM). Thus, both DBH content and DBH activity in CSF were reduced in Parkinsonian patients to less than 20% of the control values (P $ ? 0.005 ). However, the specific activity (units of enzyme activity/mg of DBH protein) in CSF of Parkinsonian patients was similar to that of control patients. These results suggest that the reduced DBH activity in CSF from Parkinsonian patients is caused by a reduction in DBH protein content, and is not due to production of an inactive form of DBH, for example, by combining with endogenous inhibitor(s). These data support our previous findings that DBH activities in the Parkinsonian brain and CSF are decreased.