Characterization and Intracellular Distribution of Pea Phytochrome I Polypeptides Expressed in E. coli

The pea phytochrome I (PI) cDNA clone, pPP1001, was expressedin E. coli. The plasmid pPP1001 contains pea PI cDNA which coversthe entire coding region with the Shine-Dalgarno consensus sequencejoined upstream of the cDNA in an expression vector pNUT6. ThepPP1001 transformants formed typical inclusion bodies when culturedat 32?C. However, when cultured at 37?C or in the presence ofisopropyl-ß-D-thiogalactopyranoside (IPTG) at 32?C,the bacteria lysed before inclusion body formation. Immuno-stainingwith anti-PI monoclonal antibody, mAP5, of transformants fixedby cold methanol showed that stainable materials were distributedin whole cytoplasmic region. When the inclusion bodies wereobserved clearly, the regions corresponding to the inclusionbodies became difficult to stain. Western blot analysis, however,showed that a ca. 100 kDa PI polypeptide was detected in thefraction from inclusion bodies and a ca. 90 kDa PI polypeptidefrom the soluble fraction. The amino acid sequence analysisof purified 100 kDa PI sample indicated that its amino terminusis blocked. However, minor signals in one experiment yieldeda sequence corresponding to the expected amino terminus of peaPI except for the initiation methionine. One of the anti-peaPI monoclonal antibodies, mAP9, that recognizes the near N-terminusof pea phytochrome was reactive to the 100 kDa polypeptide. (Received June 22, 1990; Accepted November 18, 1990)     

Photomovement in Dunaliella salina: Fluence rate-response curves and action spectra

We determined the action spectra of the photophobic responses as well as the phototactic response in Dunaliella salina (Volvocales) using both single cells and populations. The action spectra of the photophobic responses have maxima at 510 nm, the spectrum for phototaxis has a maximum at 450–460 nm. These action spectra are not compatible with the hypothesis that flavoproteins are the photoreceptor pigments, and we suggest that carotenoproteins or rhodopsins act as the photoreceptor pigments. We also conclude that the phototactic response in Dunaliella is an elementary response, quite independent of the step-up and step-down photophobic responses. We also determined the action spectra of the photoaccumulation response in populations of cells adapted to two different salt conditions. Both action spectra have a peak a 490 nm. The photoaccumulation response may be a complex response composed of the phototactic and photophobic responses. Blue or blue-green light does not elicit a photokinetic response in Dunaliella.Diagrams of the optical set-ups used for measuring the responses at the single-cell level and of the plans for building the phototaxometer described in this paper are available to the interested readerWe thank Mr. M. Kubota for a tremendous amount of technical assistance and Mr. R. Nagy for building the phototaxometer. We thank T. Kondo, Professor H. Imaseki and the members of the Laboratory of Biological Regulation, NIBB, for their help and support in various aspects of this research. This research was supported, in part, from grants from the Okazaki Large Spectrograph (Project Nos. 86-535, 87-518, 88-523), the Japanese Society for the Promotion of Science, and the College of Agriculture and Life Sciences at Cornell University to R. W.     

Absence of luminal bile increases duodenal content of cholecystokinin in rats.

The effects of the removal of bile from the proximal intestine on pancreas, plasma cholecystokinin (CCK) concentration, and duodenal content of CCK were examined in rats. Bile was excluded from the duodenum and introduced into the distal ileum through a silastic cannula for 7 days. Pancreatic juice was maintained to be normally secreted into the duodenum. After 7-day bile diversion, plasma CCK concentration and duodenal CCK content were significantly increased in bile-diverted rats. Trypsin content in the proximal intestine in bile-diverted rats was one-half that in control. Pancreatic wet weight, protein content, and DNA content in the pancreas were slightly increased, and lipase content was slightly decreased, by bile diversion, but none of these changes was statistically significant. Amylase content significantly decreased and chymotrypsin content significantly increased in bile-diverted rats. Intragastric administration of camostate (trypsin inhibitor) significantly increased plasma CCK concentration in both bile-diverted and control rats, and the net increase was much greater in bile-diverted rats than in control rats. In conclusion, bile diversion increased duodenal CCK content and increased the CCK response to luminal stimulant.     

Clostridium absonum from gas gangrene

A Clostridium perfringens-like strain was isolated from a case of gas gangrene. The morphological properties and the lecithinase reaction of the isolate were very similar to those of C. perfringens; however, the lecithinase reaction was only slightly suppressed by C. perfringens alpha-antitoxin serum and the organism was identified as Clostridium absonum from its biochemical properties.     

A monoclonal antibody that recognizes sialyl-Lea oligosaccharide, but is distinct from NS 19-9 as to epitope recognition

A murine monoclonal antibody, designated as MSW 113, was generated using a human colonic cancer cell line, SW 1116, as the immunogen. MSW 113 was shown to be directed mainly to mucin-type oligosaccharide with sialyl-Lea antigens. The reactivity of MSW 113 to sialyl-Lea was stronger than that of NS 19-9, which is believed to be raised against the same determinant group. MSW 113 binds to sialyl-Lea-ol, LS-tetrasaccharide a, and disialyllacto-N-tetraose with higher affinities, compared to NS 19-9. These two antibodies could clearly be distinguished in that MSW 113 bound to sialic acid but not to fucose, whereas NS 19-9 bound to fucose but not to sialic acid. Thus, MSW 113 is directed more toward sialic acid-containing terminal structures while NS 19-9 is directed toward fucose-containing internal structures. MSW 113 was found to be useful for detecting antigens in the bloodstream of patients, especially those with pancreas cancer. Even NS 19-9 negative patient sera were positive for MSW 113.     

Photosensitive DNA cleavage and phage inactivation by copper(II)-camptothecin

Upon irradiation with 365-nm light, copper(II)-camptothecin significantly produced single- and double-strand breaks of DNA and also induced a marked inactivation of bacteriophage. The nucleotide sequence analysis exhibited considerably random DNA cleavage. The DNA strand scission by the camptothecin-Cu(II)-UV light system, as well as the phage inactivation, was strongly suppressed by bathocuproine and catalase, indicating participation of cuprous species and hydrogen peroxide in the reaction. The present results suggest that (1) Cu(II) ion may play an important role as a cofactor in antitumor action of camptothecin and (2) the combination of copper-camptothecin plus long-wave ultraviolet light is useful against certain cancer treatment as a new photochemotherapy.     

Purification and properties of epithelial growth inhibitor (EGI) from human platelets: its separation from type beta transforming growth factor (TGF-beta)

We previously reported that sera from various kinds of animals contain a protein(s) capable of inhibiting the growth of the non-malignant epithelial cell line derived from Buffalo rat liver (BRL). In the present study, a similar epithelial cell-specific growth inhibitor (EGI) was purified to homogeneity from an acid-ethanol extract of human platelets. During purification, EGI was separated from the major component of type beta transforming growth factor (TGF-beta), which can stimulate the colony formation of the non-malignant fibroblastic cell line derived from rat kidney (NRK) in soft agar in the presence of epidermal growth factor (EGF). The purified EGI had an Mr of 27,000, and was composed of two subunits identical in Mr. It significantly inhibited the growth in monolayer cultures of three non-malignant epithelial cell lines, BRL, MDCK (from Madin-Darby canine kidney) and BSC-1 (from African green monkey kidney), at doses lower than 40 pg/ml in medium containing 10% fetal calf serum. Its inhibitory activity was stable against heating at 90 degrees C for 3 min, but not against treatment with 50 mM dithiothreitol. In addition, TGF-beta was also partially purified from the same extract. The purified TGF-beta did not show any inhibitory activity toward the growth of BRL, MDCK, BSC-1, or NRK.     

3-Methylhistidine content and pH dependency of ATPase activity of adult and embryonic chicken cardiac ventricular myosins

A distinct difference in the 3-methylhistidine (3-MeHis) content and the pH-dependency curve for calcium-activated adenosine triphosphatase (Ca-ATPase) activity was observed between chicken and mammalian cardiac ventricular myosins. The 3-MeHis content and pH dependency of the Ca-ATPase activity of myosins from adult and embryonic chicken cardiac ventricular muscles and chicken fast white and slow red muscles were almost the same.     

Retarded decline in poly-ADPR content and poly-ADPR synthetase activity in chicken dystrophic muscle

The activity of poly-ADPR synthetase declines just after hatching in normal chick muscle nuclei. However, in dystrophic chick muscle nuclei it decreases 5 weeks after hatching. A delayed decrease in the amount of poly-ADPR is also observed in dystrophic chick muscle nuclei. These observations suggest that dystrophic muscle follows an abnormal developmental program.