Characterization of rat cytochrome P-450MC synthesized in Saccharomyces cerevisiae

Rat cytochrome P-450MC cDNA was expressed in Saccharomyces cerevisiae AH22, SHY3 and NA87-11A cells under the control of the yeast ADH1 promoter and terminator. Although the three yeast strains transformed with the constructed expression plasmid, pAMC1, contained approximately three copies of the plasmid, the levels of both P-450MC mRNA and the corresponding protein in the AH22 cells carrying plasmid pAMC1 were 1.4- to 1.7-fold and 2-fold higher than in the other two strains, respectively. The P-450MC protein was purified from the microsomal fraction of AH22 cells carrying pAMC1 by a rapid purification method. The apparent molecular weight, chromatographic behavior, spectral properties, substrate specificity and immunochemical properties of the purified P-450MC protein were indistinguishable from those of rat liver P-450MC-I and P-450MC-II (Sasaki, T., et al. (1984) J. Biochem. 96, 117-126). The NH2-terminal amino acid sequence of the purified protein up to 10 residues was the same as those of P-450MC-I and P-450MC-II. In addition, HPLC analysis of the microsomal fraction of AH22 cells containing pAMC1 indicated that the synthesized P-450MC protein corresponds to P-450MC-II, but not P-450MC-I. With another purification method, we obtained the cleaved P-450MC protein which lacked the NH2-terminal 30 amino acids of intact P-450MC. The spectral properties and monooxygenase activities towards benzo(a)pyrene and 7-ethoxycoumarin of the cleaved P-450MC were nearly the same as those of intact P-450MC.     

A photosystem other than PS370 also mediates the negative phototaxis of Halobacterium halobium

Abstract It was found that a photorepellent system other than photosystem 370 (PS370) also controls the behavior of Halobacterium halobium . Both the dependence of background illumination and wavelength where the response showed maximum action distinguished that photosystem from PS370 whose photoreceptor pigment is thought to be an intermediate of s-rhodopsin (sR). A mutant strain that has no detectable activity in PS370 and in photoattractant response was isolated. This mutant strain showed the repellent response due to the new photosystem.     

Effect of high carbohydrate diet on serum gamma-glutamyl transpeptidase fraction pattern in Japanese young men

Changes in the activity of serum gamma-glutamyl transpeptidase (gamma-GTP) and the percentage of the gamma-GTP fraction in healthy young men given a high carbohydrate diet (480-636 g/day, 80% of the total energy) for 21 days were examined. Serum total gamma-GTP activity showed no significant change in four healthy young volunteers who received high carbohydrate diet for 21 days. However, the percentage of the gamma-GTP (1) fraction increased significantly (P less than 0.01) from the basal level of 55.6 +/- 4.0% to 67.6 +/- 0.9% on day 10, and then decreased to 58.4 +/- 1.4% on day 21. When the experimental diet was replaced by usual diet, the percentage of the gamma-GTP (1) fraction returned to the same level as before the experiment. It is concluded from the results that the nutrient intake affects the percentage of gamma-GTP (1), but not the total serum gamma-GTP activity.     

Quantitative detection of DNA damage in the neuronal cells of the cerebellum and cerebrum by the analysis of Feulgen hydrolysis curves

Summary Touch smears of the cerebellum and cerebrum of ageing rats were fixed with methanol, hydrolyzed with 2N HCl at various temperatures and for various periods, and stained with pararosaniline-Schiff reagent. The hydrolysis curves were determined by fluorescence cytophotometry and were computer fitted to the Bateman function to determine the kinetic parameters, the initial yield of apurinic acid or single-stranded DNA (y ₀), and the rate constants for depurination or denaturation (k ₁) and depolymerization (k ₂). The values for k ₁ (1/k ₁ is correlated with the degree of chromatin condensation) and k ₂ (which reflects the degree of DNA instability) steadily increased with age. The values for y ₀, which may indicate the degree of DNA denaturation or damage present before acid hydrolysis, also increased with age in both the cerebellum and cerebrum; however, this value was lower in the cerebellum untill 15 weeks, with the situation being reversed after 35 weeks, the cross-over time being at about 25 weeks. The values of lnk ₁ and lnk ₂ were plotted as the function of the reciprocal of the absolute temperature (T) (Arrhenius plot) for both the cerebellum and cerebrum of 15- and 74-week-old rats, and the activation energies (E) for depurination and depolymerization were calculated from the slopes. In particular, the values of E for k ₂ decreased much more quickly with age and were smaller in cerebellum. In conclusion, the degree of DNA damage and DNA instability steadily increases in both the cerebellum and cerebrum of ageing rats, and this process is much faster in the cerebellum.In honour of Prof. P. van Duijn     

Mucociliary clearance in chronic sinusitis: related human nasal clearance and in vitro bullfrog palate clearance

Nasal mucociliary clearance was measured in both healthy subjects and patients with chronic sinusitis using saccharin granule technique. Nasal mucociliary transit time (ST) was significantly slower in the patients with chronic sinusitis compared with that in controls (p less than 0.005). Nasal mucus collected from each nasal cavity was used for in vitro bullfrog palate clearance studies and compared to the in vivo nasal ST. Mucociliary clearance rate (MTR) on frog palate was 12.5 +/- 2.5 mm/min in the mucus from control subjects, 6.1 +/- 1.5 mm/min in the mucus from the patients. The difference was statistically significant (p less than 0.005). The MTR on frog palate in the patients whose nasal ST was within normal range was significantly slower than that in controls (p less than 0.005), but not significantly different from that in the patients whose nasal ST was over the normal range. These results suggest that the nasal mucous properties which decreased the mucociliary clearance on frog palate did not contribute to the mucociliary clearance of the patients who had a normal one. No significant correlation existed between MTR on frog palate and nasal ST in both control and chronic sinusitis. In chronic sinusitis patients, decelerated nasal ST was recovered significantly by normal saline nebulization compared with the value before the nebulization (p less than 0.01). None of the significant change of ST was observed in control before and after the nebulization.     

Growth Regulation in Dwarf Barley Coleoptiles by the Minor Cell Wall Components, Galactose and Mannose

Sugar compositions of cell walls of dark-grown coleoptiles from12 barley strains, 11 of which were coleoptilar semi-dwarf strains,were analyzed on days 2 and 3 after germination. Major wallcomponents of the 12 strains were arabinose, xylose, and glucosein hemicellulose and cellulose; minor components were galactoseand mannose. The sugar content of each wall component per unit length wasnot correlated to any growth parameters calculated from a logisticequation simulating coleoptile growth, but the relative contentsof galactose and mannose in relation to the total wall sugarcontent was correlated to the growth rate on day 3 and the growthcontinuing period. These facts suggest that growth of these12 barley strains in the late growth stage is regulated by theminor wall components, galactose and mannose. ¹ Dedicated to the late Professor Joji Ashida. (Received October 12, 1982; Accepted January 12, 1983)     

Scanning and transmission electron microscopy of human ejaculate spermatozoa with special reference to their abnormal forms

Summary Spermatozoa from fertile and infertile human ejaculates were observed under the scanning electron microscope. A parallel study of sections was performed by transmission electron microscope.The normal head shows under the scanning electron microscope vesicular elevations in the region of the acrosome and a smooth and rigid appearance corresponding to the postnuclear cap whose occurrence is confirmed under the transmission electron microscope. Immediately anterior to this cap a shallow furrow transverses the head. Duplicated, unusually large or small and deformed heads are found under the scanning electron microscope. Most of these abnormal heads show no surface structure suggesting an acrosome.The neck and middle piece are occasionally, though frequently in abnormal spermatozoa, covered by a cytoplasmic droplet. Otherwise, the mitochondrial sheath is recognized under the scanning electron microscope as a beaded thickening in the middle piece. The lack of mitochondria is manifested by a smooth middle piece thinner than the principal portion. Transmission electron microscopy of sections reveals various types of anomalies in the number of cores, core filaments and mitochondria embedded in the cytoplasmic droplets.Abnormalities in the principal portion of the tail such as duplication, unusual thickness and length are shown under the scanning electron microscope.The investigation indicates that scanning electron microscopy is suited for the clinical as well as cytological examination of human ejaculate spermatozoa.     

The fine structure of the mesonephros of the lamprey,Entosphenus japonicus Martens

Summary The fine structure of the mesonephric kidney of the lamprey, Entosphenus japonicus Martens, has been investigated with the electron microscope and discussed from the viewpoint of comparative morphology of the mesonephros.The structure of the capillary wall of the glomerulus essentially coincides with that of higher vertebrates, though its basement membrane is remarkably thick (300–400 m) because of a dense accumulation of fibrillar material between the endothelium and the basal lamina of epithelial cell. No obvious fenestration of the endothelial cell has been observed in the glomerulus or capillaries in any part of this organ.The kidney tubule is divided into three segments: 1. neck segment composed of ciliated cells with numerous mitochondria and glycogen particles, 2. proximal tubule composed of brush bordered cells provided with extensive pinocytotic vesicles and lysosomal granules in the apical cytoplasm and with lamellar membranes in the basal, and 3. distal tubule characterized by cells which, with their abundant mitochondria and branched tubular endoplasmic reticulum (about 500 Å diameter) with a central core, closely resemble the chloride cells in the gill filament of some teleosts. The possibility that the lamellar membranes in the proximal tubule cells correspond to basal infoldings is discussed.The extensive development of the tubular reticulum and of the mitochondria in the distal tubule cells is believed to reflect the active absorption of urine chloride in the urinary tubule of lamprey mesonephric kidney evidenced by physiologists. The proximal tubule is suggested to take a part also in the urinary transport of water and ions, as the lamellar membranes found in the cells of this portion likely correspond to the basal infoldings in more advanced forms of the kidney.The epithelial cells of the ureteric duct are characterized by granules suggesting a mucous secretion. No fine structure implying an absorptive activity in this duct has been observed.     

Intravesical treatment of bladder cancer with recombinant human interferon-β

Summary In order to examine its clinical efficacy, recombinant human interferon- (rIFN-) was instilled intravesically into 51 patients with superficial bladder cancer. Ten patients, who received intermittent intravesical instillation at a dose of (3–36) × 10⁶ U rIFN- on days 1–3 every week, showed no response. Thirty-two patients received intravesical instillation at a dose of (3–36) × 10⁶ U every day for 10–20 days. Eight patients showed partial response, indicating an efficacy rate of 25%. Nine patients received divided doses of 18 × 10⁶ U twice a day every day for 10–20 days. Six patients showed partial response, indicating an efficacy rate of 67%. This value was significantly higher than that obtained by administering divided doses. The response to intravesical instillation therapy with rIFN- varies with treatment protocol. Frequent and longer exposure to rIFN- may induce better regression of superficial bladder cancer. Six incidences of side-effects were found in five cases (9.8%): pollakiuria in one, pain on micturition in two, fever in two, and eruption in one case. All of these side-effects were slight and reversible after drug withdrawal. Laboratory tests showed only a few changes with low severity. Thus, rIFN- is potentially a new drug for instillation therapy of superficial bladder cancer, in view of the absence of adverse effects.     

Lectin histochemistry in rat thyroid tumours

The thyroid tumours and background goiterous and adenomatous lesions induced in rats with diisopropanolnitrosamine (DIPN) plus methylthiouracil (MTU), and regenerative thyroid tissues after wounding were studied by lectin histochemistry. Ten weeks after cessation of the carcinogen treatments, carcinomas invading the surrounding tissues and blood vessels (13/20) and papillary micronodules (11/20) were formed in the thyroid tissues. In general, the carcinoma lesion was solitary, and the papillary micronodules were multiple in a single thyroid gland. Among the lectins tested, Maclura pomifera (MPA) and Solanum tuberosum (STA) showed specific binding with both carcinoma and papillary micronodule lesions, but not with the background goiterous and adenomatous lesions and regenerative thyroid tissues. The former both lesions showed higher labelling indices with BUdR or 3H-thymidine and poorer thyroglobulin accumulation than the latters, thereby indicating their enhanced proliferative capability and depressed potency of cyto-differentiation. The common cytological and histochemical properties of carcinoma lesions and papillary micronodules allow us to regard the latter as pre-invading carcinoma lesions. The lectins MPA and STA may be, therefore, used as the specific markers of malignancy in rat thyroid carcinogenesis.