Mitochondrial DNA haplogroups and age at onset of Machado-Joseph disease/spinocerebellar ataxia type 3: a study in patients from multiple populations.

BACKGROUND AND PURPOSE: Mitochondrial dysfunction has been implicated in the pathogenesis of several neurodegenerative disorders, including Machado-Joseph disease (MJD), an autosomal dominant late-onset polyglutamine ataxia that results from an unstable expansion of a CAG tract in the ATXN3 gene. The size of the CAG tract only partially explains age at onset (AO), highlighting the existence of disease modifiers. Mitochondrial DNA (mtDNA) haplogroups have been associated with clinical presentation in other polyglutamine disorders, constituting potential modifiers of MJD phenotype. METHODS: A cross-sectional study, using 235 unrelated patients from Portugal, Brazil, India and Japan, was performed to investigate if mtDNA haplogroups contribute to AO of MJD. mtDNA haplogroups were obtained after sequencing the mtDNA hypervariable region I. Patients were classified in 15 phylogenetically related haplogroup clusters. RESULTS: The AO was significantly different among populations, implying the existence of other non-CAG factors, which seem to be population specific. In the Portuguese population, patients classified as belonging to haplogroup JT presented the earliest onset (estimated onset 34.6 years of age). Haplogroups W and X seem to have a protective effect, causing a delay in onset (estimated onset 47 years of age). No significant association between haplogroup clusters and AO was detected in the other populations or when all patients were pooled. Although haplogroup JT has already been implicated in other neurodegenerative disorders, no previous reports of an association between haplogroups W and X and disease were found. CONCLUSIONS: These findings suggest that haplogroups JT, W and X modify AO in MJD. Replication studies should be performed in European populations, where the frequency of the candidate modifiers is similar.

Effect of low-concentration chlorine dioxide gas against bacteria and viruses on a glass surface in wet environments.

AIMS: To evaluate the efficacy of low-concentration chlorine dioxide (ClO(2)) gas against model microbes in the wet state on a glass surface. METHODS AND RESULTS: We set up a test room (39 m(3)) and the ClO(2) gas was produced by a ClO(2) gas generator that continuously releases a constant low-concentration ClO(2) gas. Influenza A virus (Flu-A), feline calicivirus (FCV), Staphylococcus aureus and Escherichia coli were chosen as the model microbes. The low-concentration ClO(2) gas (mean 0.05 ppmv, 0.14 mg m(-3)) inactivated Flu-A and E. coli (>5 log(10) reductions) and FCV and S. aureus (>2 log(10) reductions) in the wet state on glass dishes within 5 h. CONCLUSIONS: The treatment of wet environments in the presence of human activity such as kitchens and bathrooms with the low-concentration ClO(2) gas would be useful for reducing the risk of infection by bacteria and viruses residing on the environmental hard surfaces without adverse effects. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that the low-concentration ClO(2) gas (mean 0.05 ppmv) inactivates various kinds of microbes such as Gram-positive and Gram-negative bacteria, enveloped and nonenveloped viruses in the wet state.

Chlorine dioxide is a more potent antiviral agent against SARS-CoV-2 than sodium hypochlorite.

BACKGROUND: A new coronavirus (SARS-CoV-2) abruptly emerged in Wuhan, China, in 2019 and rapidly spread globally to cause the COVID-19 pandemic. AIM: To examine the anti-SARS-CoV-2 activity of the potent disinfectant Cleverin, the major disinfecting component of which is chlorine dioxide (ClO2); and to compare the results with that of sodium hypochlorite in the presence or absence of 0.5% or 1.0% foetal bovine serum (FBS). METHODS: Concentrated SARS-CoV-2 viruses were treated with various concentrations of ClO2 and sodium hypochlorite and 50% tissue culture infective dose was calcurated to evaluate the antiviral activity of each chemical. FINDINGS: When SARS-CoV-2 viruses were treated with 0.8 ppm ClO2 or sodium hypochlorite, viral titre was decreased only by 1 log10 TCID50/mL in 3 min. However, the viral titre was decreased by more than 4 log10 TCID50/mL when treated with 80 ppm of each chemical for 10 s regardless of presence or absence of FBS. It should be emphasized that treatment with 24 ppm of ClO2 inactivated more than 99.99% SARS-CoV-2 within 10 s or 99.99% SARS-CoV-2 in 1 min in the presence of 0.5% or 1.0% FBS, respectively. By contrast, 24 ppm of sodium hypochlorite inactivated only 99% or 90% SARS-CoV-2 in 3 min under similar conditions. Notably, except for ClO2, the other components of Cleverin such as sodium chlorite, decaglycerol monolaurate, and silicone showed no significant antiviral activity. CONCLUSION: Altogether, the results strongly suggest that although ClO2 and sodium hypochlorite are strong antiviral agents in absence of organic matter but in presence of organic matter, ClO2 is a more potent antiviral agent against SARS-CoV-2 than sodium hypochlorite.

Elongation of repetitive DNA by DNA polymerase from a hyperthermophilic bacterium Thermus thermophilus.

Short repetitive DNA sequences are believed to be one of the primordial genetic elements that served as a source of complex large DNA found in the genome of modern organisms. However, the mechanism of its expansion (increase in repeat number) during the course of evolution is unclear. We demonstrate that the DNA polymerase of the hyperthermophilic bacterium Thermus thermophilus can elongate oligoDNA with several tandem repeats to very long DNA in vitro. For instance, 48mer repetitive oligoDNA (TACATGTA)(6), which has 25% GC content and a palindromic sequence, can be elongated up to approximately 10 000 bases by DNA polymerase at 74 degrees C without template DNA. OligoDNA having a different GC content or a quasi-palindromic sequence can also be elongated, but less efficiently. A spectroscopic thermal melting experiment with the oligoDNA showed that its hairpin-coil transition temperature was very close to the elongation reaction temperature (74 degrees C), but was much higher than the temperature at which duplex oligoDNA can exist stably. Taken together, we conclude that repetitive oligoDNA with a palindromic or quasi-palindromic sequence is elongated extensively by a hyperthermophilic DNA polymerase through hairpin-coil transitions. We propose that such an elongation mechanism might have been a driving force to expand primordial short DNA.

Establishment of tumor cell lines as an independent prognostic factor for survival time in patients with small-cell lung cancer.

We studied tumor samples from 39 patients, who entered our study from January 1989 to May 1990, to assess whether the ability to establish a continually growing tumor cell line from fresh tumor specimens can be associated with decreased survival times in patients with small-cell lung cancer. The tumor samples were used to establish cell lines in culture using a serum-free medium supplemented with hydrocortisone, insulin, transferrin, estrogen, and selenium (HITES). Thirty-three of these specimens were obtained by fiberoptic bronchoscopy from primary sites during routine diagnostic procedures. A total of 11 (28%) cell lines were established: seven (21%) from 33 primary tumors and four (80%) from five peripheral lymph nodes. Survival times of the 11 patients whose tumor cell specimens continually grew in culture at any time during their clinical course were significantly shorter than those of the 28 patients whose tumor cell specimens did not grow in vitro (median survival time of 26 weeks versus 73 weeks; P = .0068). Cox's proportional hazards model, including sex, age, Eastern Cooperative Oncology Group performance status, stage, source of specimen, treatment, and in vitro tumor cell growth in the overall patient group, showed that cell line establishment (P = .0017) and no therapy (P = .0015) were the most important factors indicating poor survival time. For the subgroup of 23 primary tumor patients, the important factors (in decreasing order) that indicated decreased survival times were the establishment of a cell line (P = .0112) and with cyclophosphamide-doxorubicin-vincristine alternating with cisplatin-etoposide, versus cisplatin-vincristine-doxorubicin-etoposide therapy (P = .0463). Our study demonstrates that in vitro tumor cell growth is an adverse predominant prognostic factor in patients with small-cell lung cancer.

Purification and characterization of D-3-aminoisobutyrate-pyruvate aminotransferase from rat liver.

D-3-Aminoisobutyrate-pyruvate aminotransferase (EC 2.6.1.40) was purified 1900-fold from rat liver extract. The purified enzyme showed a molecular mass of 180 kDa by gel-permeation HPLC analysis using a TSK gel G3000SW column. Reductive polyacrylamide gel electrophoresis in sodium dodecyl sulfate resulted in identification of a single band of approx. 50 kDa, indicating that the native enzyme is probably a tetrametric protein. The specific activity of the purified enzyme was 1.14 mumol/min per mg protein. D-3-Aminoisobutyrate and beta-alanine were good amino donors. The Km value for L-3-aminoisobutyrate was 100-times larger than that for the D-isomer. The apparent Km values for D-3-aminoisobutyrate and beta-alanine were 35 and 282 microM, respectively. Pyruvate, glyoxylate, oxalacetate, 2-oxo-n-valerate, and 2-oxo-n-butyrate were good amino acceptors. The apparent Km values for pyruvate and glyoxylate were 32 and 44 microM, respectively.

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.

Lack of an association between cystatin C gene polymorphisms in Japanese patients with Alzheimer's disease.

Associations between polymorphisms of the cystatin C gene (CST3) at 5' flanking region and exon 1 in Caucasian patients with late onset AD and exon 1 in a US study of late onset AD have been reported. Clinically diagnosed Japanese patients with AD and Japanese normal control subjects were assessed for the presence of polymorphisms of CST3. The authors could not confirm the previously reported association between CST3 polymorphisms and AD in Japan. Age had no effect on the CST3 genotype.

Preoperative histologic diagnosis of chest wall invasion by lung cancer using ultrasonically guided biopsy.

Before operation, we evaluated the usefulness of ultrasonically guided needle biopsy to detect histologically invasion of the chest wall by tumor in patients with lung cancer. Ultrasonically guided needle biopsy, computed tomography, and ultrasonography were done in 29 patients with lung cancer. In all of them, chest wall invasion was histologically confirmed by thoracotomy. As for the diagnosis of chest wall invasion, sensitivity, specificity, and accuracy of ultrasonically guided needle biopsy diagnosis were 61.5%, 100%, and 82.8%, respectively; for diagnosis by computed tomographic scan these figures were 69.2%, 75.0%, and 72.4%, respectively; and for diagnosis by ultrasonography they were 76.9%, 68.8%, and 72.4%, respectively. We had no false-positive cases in the ultrasonically guided needle biopsy assessment and no fatal complications or implantation metastases. Our results indicate that ultrasonically guided needle biopsy is safe and useful for preoperative histologic diagnosis of chest wall invasion in cases in which combined chest wall resection is being considered in patients with lung cancer.

Intraoperative pleural lavage cytology in lung cancer patients.

Cytology of intraoperative pleural lavage was examined in 164 lung cancer patients who underwent pulmonary resections. None of the patients had any pleural effusion or dissemination. Cytology was performed three times: (1) at thoracotomy, (2) immediately after resection, and (3) after washing the pleural cavity with 5,000 mL of physiological saline solution just before closure of the chest wall. Twenty-three patients (14%) had more than one positive cytological finding. The frequency of positive cytological findings was significantly correlated with pathological T classification, pleural status, and pathological stage. The positive lavage group had a significantly higher recurrence rate than the negative lavage group in patients with stage I or stage II cancer. Four patients in the positive lavage group (17.4%) had recurrence in pleura or pericardium whereas only 1 patient in the negative lavage group (0.7%) had a recurrence in pericardium. The positive cytological finding of pleural lavage has more important meaning as a prognostic factor in stage I and stage II and indicates a greater possibility of recurrence in pleura or pericardium, but further examinations to evaluate the viability of detected malignant cells are required so that the positive cytological findings of pleural lavage can be regarded as subclinical pleural dissemination.

Genetic studies in Parkinson's disease with an alpha-synuclein/NACP gene polymorphism in Japan.

Dinucleotide repeat polymorphism has been observed in the promoter of the alpha-synuclein (alpha-SYN)/NAC precursor protein (NACP) gene. Alpha-SYN/NACP allele 3 (described by Xia et al. (Ann. Neurol., 40 (1996) 207), equivalent to allele 1 described by Krüger et al. (Ann. Neurol. 45 (1999) 611) is reported to be significantly more frequent among patients with sporadic Parkinson's disease (sPD) than controls. In this study, we genotyped the same alpha-SYN/NACP polymorphism in Japanese sPD patients and healthy controls, but found that any aliele showed no significant difference between the two groups.

N-acetyl-L-aspartic acid, N-acetyl-alpha-L-aspartyl-L-glutamic acid and beta-citryl-L-glutamic acid in human urine.

N-Acetyl-L-aspartic acid (NA-Asp), N-acetyl-alpha-L-aspartyl-L-glutamic acid (NA-Asp-Glu) and beta-citryl-L-glutamic acid (beta-CG), which are known to occur in the brain, have been isolated from human urine. Their identities were proved by comparing them with synthetic NA-Asp, NA-Asp-Glu and beta-CG using electrophoretic and chromatographic methods and by acid hydrolysis. A method was developed for the quantitation of NA-Asp, NA-Asp-Glu and beta-CG in human urine. It consists of ion-exchange chromatography followed by gas-chromatographic analysis. The amounts of urinary excretion of NA-Asp, NA-Asp-Glu and beta-CG were 41.2 +/- 10.1 (n = 27), 20.8 +/- 9.6 (n = 27) and 30.2 +/- 13.2 (n = 21) mumol/g creatinine in adult males, and 62.2 +/- 16.3 (n = 27), 24.0 +/- 8.2 (n = 27) and 40.5 +/- 21.1 (n = 24) mumol/g creatinine in adult females, respectively.

Touch imprint cytology and immunohistochemistry for the assessment of sentinel lymph nodes in patients with breast cancer.

AIMS: Accurate evaluation of sentinel nodes is of clinical importance to avoid further surgery for axillary node dissection. A prospective study was carried out to investigate the feasibility and accuracy of touch imprint cytology (TIC) and touch imprint immunohistochemistry (TIHC). METHODS: Two hundred and five sentinel nodes from consecutive 118 patients with primary breast cancer were studied after successful identification of sentinel nodes. Sentinel nodes were sectioned at 2 mm intervals and imprint specimens prepared from all cut surfaces were subjected to Papanicolaou staining and immunohistochemical staining using anti-cytokeratin antibody. RESULTS: Forty-nine sentinel nodes from 40 patients were positive by permanent section. The sensitivity of TIC was 84% (41/49) per sentinel node and 83% (33/40) on a per patient basis. The sensitivity of TIHC was 86% (42/49) per sentinel node and 83% (33/40) on a per patient basis. When the results of TIC and TIHC were combined, the sensitivity was 88% (43/49) per sentinel node and 85% (34/40) on a per patient basis. Among the 156 negative sentinel nodes, four sentinel nodes from four different patients were consistently positive by TIC and TIHC, but only one patient out of 78 node-negative patients was upstaged. CONCLUSIONS: Touch imprint cytology is sufficiently sensitive for intraoperative evaluation of sentinel nodes. A slight improvement in the sensitivity is expected when immunohistochemistry is used. The combination of these methods provides better sensitivity than either method alone.

Pleomorphic adenoma in the periphery of the lung. Report of a case and review of the literature.

A pleomorphic adenoma of the lung recurred after 9 years. The primary tumor consisted mainly of cartilaginous and fibrous elements with a small area of epithelial cell nests, whereas the second one possessed epithelial cell nests with cartilaginous stroma. Immunohistochemical studies showed that both tumors had neoplastic cells with immunoreactive S100, keratin, actin, vimentin, and glial fibrillary acid protein-positive cytoplasm. The primary tumor, which was resected from the periphery of the lung, was not connected with the trachea or the bronchus macroscopically. To our knowledge, the literature contains only six reports of pleomorphic adenoma in the lung.

Evidence for other binding component(s) than retinoid receptors specifically interacting with a retinoic acid response element in the rat liver.

A nuclear component interacting with a retinoic acid response element (RARE-beta) derived from the all-trans-retinoic acid receptor beta (RAR beta) gene promoter was detected in the rat liver by gel-shift assay. Competition experiments using the competitors of the other retinoid response elements and non-specific sequences upon the binding of this hepatic component (HBC) to the RARE-beta revealed that the binding of the HBC to the RARE-beta was competed only by the self competitor, but not the other elements, suggesting that the HBC is specific for the RARE-beta. Moreover, although the specific monoclonal antibodies for RAR alpha, RAR beta, and RAR gamma reacted and shifted up the DNA complexes of endogenous as well as recombinant RARs, the DNA complex with HBC was not subjected to the immunoreaction with the antibodies. Thus, newly identified HBC, which is distinct from the characterized retinoid receptors, may modulate retinoid signaling through its direct binding upon the RARE-beta.

[An autopsy case of Degos' disease with ascending thoracic myelopathy].

Degos' disease is a rare multisystem vasculopathy of unknown etiology. We report a 44-year-old man who presented himself with gait and sensory disturbances mainly due to thoracic transverse myelopathy four years after the appearance of many characteristic umbilicated papules over the trunk and extremities. He did not complain of abdominal pain or discomfort. Laboratory, electrophysiological and imaging studies did not show any characteristic change, except for the increase of protein contents and cell counts in the cerebrospinal fluid. We tried methylprednisolone pulse-dose therapy (1,000 mg/day x 3 days) five times, but this patient's neurological condition worsened stepwise after it, although the appearance of new skin lesion was suppressed. Intravenous infusion of ozagrel sodium and cyclophosphamide (1,000 mg/day) were also ineffective, and this patient died of respiratory failure after showing oculomotor paresis and comatose state. Necropsy revealed Degos' disease-specific vasculopathy in the central nervous system and the gastrointestinal tract, where occlusions of small-sized arteries and veins due to the intimal thickening were evident. The tissue necrosis was macroscopically remarkable in the brainstem and the thoracic spinal cord. The efficacy of steroid therapy for this disease should be investigated more carefully.

[General pharmacology of (2"R)-4'-O-tetrahydropyranyladriamycin, a new antitumor antibiotic].

General pharmacology of (2"R)-4'-O-tetrahydropyranyladriamycin hydrochloride (THP) was studied in experimental animals. Intravenous administration of THP showed no significant effect on the respiratory and cardiovascular systems, such as blood pressure, heart rate, ECG and respiration in anesthetized rabbits and dogs. But in rats and cats, THP produced a transient decrease in blood pressure resulted from vasodilation. The hypotension was not inhibited by antihistaminics. Contraction of isolated guinea-pig atria was stimulated by THP at high concentrations (10(-4) g/ml). THP inhibited the spontaneous movement of isolated rabbit ileum and rat uterus (virgin and pregnant) at high concentrations (10(-4) g/ml). In some isolated guinea-pig ileum preparations, THP partially (6 approximately 36%) antagonized the contraction inducing by acetylcholine, histamine, serotonin and barium chloride. Urine volume and urinary excretion of electrolytes were increased by intravenous injection of 5.0 mg/kg THP. Vascular permeability was progressed when administered intracutaneously. Hemolytic effect was shown at high concentrations (10(-4) g/ml) but no effect on the coagulation was found. No significant effect of THP was observed on the general behavior and central nervous system, autonomic nervous and peripheral nervous systems. Also, THP had no significant effect on gastrointestinal propulsion in mice, the mucous membranes of the stomach and duodenum of rats, or gastric acid and bile secretion in rats.

[Study on local recurrence of squamous cell carcinoma of the lung on hilar lesion].

The study population consisted of 62 patients with squamous cell carcinoma of the lung on hilar lesion who underwent curative or relative curative resection during the seven year period between January, 1980 and December, 1986. We studied the correlation between local recurrence and the distance from the surgical margin of the trachea or bronchus to the tumor. 1) As the classification of the T and N factors increased, the incidence of local recurrence became higher. 2) In order to prevent local relapse, we need to completely resect the hilar and mediastinal lymph nodes, and we must maintain a distance of 16 mm or more between the surgical margin of the trachea or bronchus and the tumor.

[An elderly case of chronic inflammatory demyelinating polyneuropathy with acute onset in the course of diabetes mellitus].

A 77-year-old man was admitted because of muscle weakness in both upper and lower extremities. Diabetes mellitus was diagnosed in 1988 and he had been treated by oral hypoglycemic agents. He had a common cold at the end of January, 1997. Muscle weakness appeared in the upper extremities, followed by the lower extremities at the end of February. No sensory disturbance or dysuria was recognized. Nerve conduction study revealed distally dominant demyelinating polyneuropathy. Guillain-Barré's syndrome was diagnosed and he recovered completely following immunological absorption therapy (IAT). However, he had quadriplegia again at the end of April. He was treated by IAT combined with corticosteroid and has shown no relapse. In June, 1997, gastric cancer was detected by upper gastrointestinal fiberscopy and subtotal gastrectomy was performed. Judging from this clinical course, this case seems to be chronic inflammatory demyelinating polyneuropathy (CIDP) with acute onset. Many kinds of causes often contribute to the pathogenesis of neuropathy in the elderly. So in cases of progression or worsening, we should consider such possibilities and it is necessary not to exclude treatable causes of neuropathy.