A ubiquitin-conjugating enzyme in fission yeast that is essential for the onset of anaphase in mitosis.

A cDNA encoding a ubiquitin-conjugating enzyme designated UbcP4 in fission yeast was isolated. Disruption of its genomic gene revealed that it was essential for cell viability. In vivo depletion of the UbcP4 protein demonstrated that it was necessary for cell cycle progression at two phases, G2/M and metaphase/anaphase transitions. The G2 arrest of UbcP4-depleted cells was dependent upon chk1, which mediates checkpoint pathway. UbcP4-depleted cells arrested at metaphase had condensed chromosomes but were defective in separation. However, septum formation and cytokinesis were not restrained during the metaphase arrest. Overexpression of UbcP4 specifically rescued the growth defect of cut9ts cells at a restrictive temperature. cut9 encodes a component of the anaphase-promoting complex (APC) which is required for chromosome segregation at anaphase and moreover is defined as cyclin-specific ubiquitin ligase. Cdc13, a mitotic cyclin in fission yeast, was accumulated in the UbcP4-depleted cells. These results strongly suggested that UbcP4 is a ubiquitin-conjugating enzyme working in conjunction with APC and mediates the ubiquitin pathway for degradation of "sister chromatid holding protein(s)" at the onset of anaphase and possibly of mitotic cyclin at the exit of mitosis.

Covalent modification of all members of human cullin family proteins by NEDD8.

Recently we found that NEDD8, a ubiquitin-like protein, was linked covalently to human cullin-4A (abbreviated Cul-4A) by a new ubiquitin-related pathway that is analogous to but distinct from the ligating system for SUMO1, another ubiquitin-like protein. However, it remained unknown whether the other five members of the family of human cullin/Cdc53 proteins are modified by NEDD8. Here we report that all Hs-Cul family proteins, such as Cul-1, Cul-2, Cul-3, Cul-4B, and Cul-5, in addition to Cul-4A, were modified by covalent attachment of NEDD8 in rabbit reticulocyte lysates. Moreover, by comprehensive Northern-blot analyses, we examined multiple tissue distributions of the messages for all Cul-family proteins, NEDD8, and the NEDD8-ligating system consisting of APP-BP1/hUba3, and hUbc12, which function as E1- and E2-like enzymes, respectively. The expressions of Cul-1, Cul-2, and Cul-3 resembled each other and were apparently correlated to those of NEDD8 and the NEDD8-ligating system in various human cells and tissues. However, the mRNA levels of Cul-4A, Cul-4B, and Cul-5 differed considerably from each other as well as from other Cul-family proteins. The enhanced expression of all Cul-family proteins except Cul-5 was observed in a variety of tumor cell lines.

Molecular cloning and sequence analysis of cDNAs for five major subunits of human proteasomes (multi-catalytic proteinase complexes).

Proteasomes are multicatalytic proteinase complexes consisting of a set of non-identical polypeptide components. Of these multiple components, the nucleotide sequences of five major subunits (named HC2, HC3, HC5, HC8 and HC9) of human proteasomes have been determined from recombinant cDNA clones by screening a human HepG2 hepatoblastoma cell cDNA library with rat proteasome cDNAs isolated previously as probes. The polypeptides deduced from their nucleotide sequences consisted of 263, 234, 241, 255 and 261 amino acid residues with calculated molecular weights of 29,554, 25,897, 26,487, 28,431 and 29,482, respectively, which are encoded by single independent genes. The primary structures of these subunits of human proteasomes closely resemble those of their rat counterparts and show considerably high inter-subunit homology, although the homology of HC5 is relatively low. These findings, together with the structural similarities of other eukaryotic proteasomes including those of Drosophila and yeast (Saccharomyces cerevisiae) support and extend the previously proposed concept that eukaryotic proteasome genes form a multi-gene family with the same evolutionary origin.

Isolation and characterization of alpha-type HC3 and beta-type HC5 subunit genes of human proteasomes.

Eukaryotic proteasomes from an evolutionarily conserved multi-gene family and are thought to have originated from a common ancestral gene and diverged into alpha-type and beta-type subgroups. To understand the molecular basis of the proteasome genes, we isolated and characterized two human proteasome genes econding the alpha-type HC3 and beta-type HC5 subunit. The functional genes for HC3 and HC5 are similar in being approximately 15 kb in length, but differ in having exon numbers of 9 and 6, respectively. Analyses of about 2.5 to 3.0 kb of the 5'-flanking regions of these two genes revealed the absence of TATA and CAAT promoter elements. However, two or three GC boxes were found. By analysis of the transcriptional regulatory activities in the 5'-flanking regions of the two genes, these GC boxes were found to function coordinately as promoters of the two genes. Interestingly, the HC3 gene possesses an additional silencer element in the 5'-upstream region near the first exon. This element is also able to repress the promoter activities of other genes, such as the HC5 and the type 1 glucose transporter genes, irrespective of whether it has a sense or antisense orientation, indicating that it acts as a general transcriptional silencer. The HC5 gene does not have this silence element, and its promoter activity is five to ten times that of HC3. These results show that the human proteasomal HC3 and HC5 genes differ not only in their genomic structures, such as their numbers of exons and their exon-intron organizations, but also in the mechanisms regulating their transcription, suggesting that they diverged at an early stage of evolution.

Cloning and expression of cDNA encoding a human ubiquitin-conjugating enzyme similar to the Drosophila bendless gene product.

cDNA encoding a novel human ubiquitin-conjugating enzyme has been cloned from an epidermoid carcinoma KB cDNA library. This clone encodes a protein of 152 amino acids with a calculated M(r) of 17,137. The amino acid sequence showed 80% identity with the Drosophila's bendless gene product (ubiquitin-conjugating enzyme E2). The corresponding transcripts are highly expressed in heart, skeletal muscle, and testis. The product expressed in Escherichia coli exhibited the ability to form a thiol ester linkage with ubiquitin in a ubiquitin-activating enzyme E1-dependent manner. These results suggest that the obtained cDNA encodes a novel human E2 which may be involved in protein degradation mainly in the muscles and testis.

Personal exposure and health effect relationship for NO2 with urinary hydroxyproline to creatinine ratio as indicator.

Establishment of an exposure-effect relationship was attempted between personal nitrogen dioxide (NO2) exposure and urinary hydroxyproline to creatinine ratio of approximately 800 adult women, who were mothers of primary schoolchildren living in two communities around Tokyo. Daily average of the personal NO2 exposure (ENO2) was measured during wintertime by a newly developed personal monitor exposed for 24 hours. The hydroxyproline to creatinine ratio (HOP:C) in the urine sample collected early in the morning of the day for ENO2 measurement was used as a biochemical indicator of the health effect of NO2 exposure. The HOP:C was found to have significant correlation with ENO2 and number of cigarettes smoked actively and passively. ENO2, however, had no correlation with the intensity of the smoking levels; they might affect HOP:C independently. Stepwise multiple regression analysis revealed that HOP:C could be predicted by ENO2 and smoking habits at a high confidence level. The regression analysis of the active smokers' group indicated that a few cigarettes was enough to increase the HOP:C, while in the case of passive smoking, HOP:C increased proportionally to the number of cigarettes.

Detection of fowl mites inside two hospital rooms.

Many mites were found on bookshelves near the windows in 2 rooms of the Department of Pediatrics on the 4th floor in the Tokai University Hospital. Fortunately, nobody reported being bitten by, or experiencing itching due to, the mites. The arthropods were tentatively identified as adults of the northern fowl mite, Ornithonyssus sylviarum (Canestrini et Frazago, 1877), commonly called "Torisashi-dani" in Japanese. It appears the mites came from an empty wagtail nest which was outside the windows. Since migrating birds like the wagtail may carry O. sylviarum and other pests and pathogens, and their possible transfer to domestic birds, more attention should be paid to the danger posed by bird mites.

An improved method for analysis of urinary hydroxyproline by an automated analyzer.

An improved method of analysis has been devised for estimating the amount of hydroxyproline excreted into the urine. This study has been performed to overcome the difficulties accompaning mass-examinations which are indispensable in epidimiological studies. Urine samples are pretreated with a resin-charcoal mixture for elimination of interfering substances or color inhibitions, and the supernatant is treated by evaporation to avoid complications followed by pH adjustment. The residue is reconstituted with diluted HCL. After pretreatment, samples a analyzed with an automated analyzer. Linearity of standard calibration curve, and recovery and reproducibility of this assay are satisfactory. This simple procedure of analysis requires little time. This method is also useful for routine clinical examinations.

Relationship between housing environment and allergic symptoms of children using ATS-DLD questionnaires.

Using pupils in three areas with different environmental factors (Suginami-ku, Tokyo: Aikawa-machi, Kanagawa prefecture: and Shimizu-shi, Shizuoka prefecture) as subjects, respiratory symptoms and domestic environment were surveyed by meams of the American Thoratic Society-Division of Lung Disease (ATS-DLD) questionnaires together with measurement of total immunoglobulin E (IgE) by the radioimmunosorbent test (RIST) and specific mite and cedar IgE by the radioallergosorbent test (RAST), as well as the relationship between "allergic symptoms" as designated in the ATS-DLD questionnaire and housing structure. The following knowledge was obtained. According to the ATS-DLD standardized questionnaires for prevalence of respiratory symptoms and allergic symptoms, Aikawa showed a significantly low value of 8.5% (p less than 0.001) compared with 15.8% for Suginami and 15.7% for Shimizu, referring to allergic rhinitis only. The positive rate of 300 U/ml and over of total IgE was 55.5% in Suginami, 49.1% in Shimizu and 40.4% in Aikawa. Aikawa showed the lowest the positive rate of total IgE in allergic symptoms. The housing was classified into three types according to the original structure each of which had characteristic ventilation rates. The positive rates according to the housing structure showed no differences by areas for Type A (wooden house) with good ventilation, but for Types B (mortar frame house) and C (steel reinforced frame house) with airtight structures, the rates increased in the order of Aikawa less than Shimizu less than Suginami, and a significant difference was observed between Aikawa and Suginami (p less than 0.01). However, in the same area no difference was noted between Type A and Types B and C. No difference in the positive rate of allergic experiences by housing structure, was noted among areas and housing structures in the same area for the group not experiencing allergic symptoms (A. Inexp. Group), while no difference was noted for Type A but the rates were high in the order of 60.0% in Aikawa, 65.2% in Shimizu and 75.3% in Suginami for Types B and C in the group experiencing allergic symptoms (A. Exp. Group). Furthermore, in the same area when the Type A was compared with Types B and C, the latter showed a higher positive rate than the formar in all three areas, with a significant difference (p less than 0.05) noted in Suginami.

Epidemiological study on the hydroxyproline: creatinine ratio as an index of growth rate.

A number of studies has suggested that urinary hydroxyproline can be used as an index of collagen metabolism and urinary hydroxyproline excretion is closely related to growth hormone. This report is a cohort study to demonstrate the relationship between the urinary hydroxyproline: creatinine ratio and growth rate for four years. The pattern of increased urinary hydroxyproline: creatinine ratio in adolescence appeared to be closely ralated to the general pattern of growth rate measured in school children. In has been shown that the urinary hydroxyproline: creatinine ratio is the most suitable index of the growth rate.

Personal exposure to nitrogen dioxide and its relation to indoor and outdoor air quality.

An estimating equation of annual geometric average of personal NO2 exposure was proposed. The average could be calculated from three variables, that is, personal NO2 exposures in one winter and one summer month and ratio of winter period in a year. Ten housewives living in Suginami Ward, a residential area in Tokyo, were chosen as subjects of this study. They were asked to measure daily averages of the personal NO2 exposures with a filter badge. Measurements were performed on consecutive seven days in every month from January to December of 1982 for a total of 84 samples per person. Their activities, the amount of fuel consumption and other living conditions were recorded at the same time. Outdoor NO2 concentrations and maximum, minimum and average temperatures were also measured at the center of their living area. The personal NO2 exposure and its seasonal variation were found to depend mainly on the type of space heaters, frequency of ventilation fan usage in a kitchen, and the amount of fuel consumption.

A comparative study on the health effects of smoking and indoor air pollution in summer and winter.

This study was performed by a comparison between summer and winter for the purpose of demonstration the actual conditions of health effects of indoor air pollution with special reference to NO2 and smoking, on the subjects composed of 820 school children and their 546 mothers in the two areas with different ambient NO2 concentrations. In either case, examination was carried out with standardized questionnaire test for respiratory symptoms, personal NO2 exposure measurement using the filter badge by Yanagisawa, and analysis of urinary hydroxyproline and creatinine in two areas with different ambient NO2 levels. Personal NO2 exposure level in winter season was 2-3 times higher than that in summer, particularly NO2 level among residents living in homes with non-vented stove for space heating was substantially higher from those of residents with vented stove. Wives with vented stove had a moderate exposure level in winter season by the contribution of NO2 originated from the kitchen and poor ventilation rate. Since the hydroxyproline to creatinine ratio (HOP-ratio) of children increased more, their household location were nearer to any heavy traffic roads in summer, health effects from automobile exhaust were suggested only in summer season. In summer season, personal NO2 exposure level were almost the same with the ambient NO2 concentrations over both areas. These results suggest that indoor air pollution in winter season may be separated from outdoor air pollution. It was a matter of course that hydroxyproline to creatinine ratio in winter season was higher than that in summer, in any group and in any area, but the range of variation of hydroxyproline to creatinine ratio was smaller by far than that of personal NO2 exposure level. Judging from urinary hydroxyproline to creatinine ratio, health effects of active smoking and passive smoking increased with increasing the number of smoked, dose-dependently in any season. According to stepwise multiple regression analysis, hydroxyproline to creatinine ratio had significant relation to either NO2 and active or passive smoking, but personal NO2 exposure level had no relation to cigarette smoking. NO2 and cigarette smoke were seemed to make hydroxyproline to creatinine ratio increase, independently each other.

Adenocystic carcinoma in computed tomography.

Computed tomography was introduced in 1972 by Ambrose and Hounsified in England. At first it was designed for diagnostic radiology and neurosurgery of the brain and skull. Soon, in 1974, whole-body computerized tomography scanners went into operation in the United States and then came into worldwide use. The use of computed tomography in maxillofacial regions is rare. In the report two adenocystic carcinomas in the maxilla are described by computed tomography, and the images thus produced are compared to those obtained by ordinary tomography and isotope scintigraphy.

Exposure to ozone and erythrocyte osmotic resistance in the rat.

In order to learn the biological effect of photochemical oxidants on living bodies, we exposed newborn and adult rats, of both sexes, to ozone at a concentration of 0.25 ppm, which can be encountered in an urban environment, and then measured the osmotic resistance of their erythrocytes. The results of experiments using newborn rats indicated a positive increase in the osmotic resistance of erythrocytes in whole blood following ozone exposure for 4 weeks. An increase in the osmotic resistance of erythrocytes in the top part obtained by centrifugation was observed following ozone exposure for 12 weeks. This tendency was especially evident among male rats. On the other hand, no increase in the osmotic resistance of erythrocytes was recognized in the adult animals which had been exposed to the same concentration of ozone for 18 months.