Carbonic Anhydrase from the Blue-Green Alga (Cyanobacterium) Anabaena variabilis

Carbonic anhydrase (CA) activity was detected in homogenatesfrom Anabaena variabilis ATCC 29413, M-2 and M-3, but not inthe suspension of the intact cells. Activity was higher in cellsgrown in ordinary air (low-CO₂ cells) than in those grown inair enriched with 2–4% CO₂ (high-CO₂ cells). Fractionationby centrifugation indicated that the CA from A. variabilis ATCC29413 is soluble, whereas both soluble and insoluble forms existin A. variabilis M-2 and M-3. The addition of dithiothreitoland Mg^{2 $} greatly decreased the CA activity of A. variabilisATCC 29413. The specific activity of the CA from A. variabilis ATCC 29413was increased ca. 200 times by purification with ammonium sulfate,DEAE-Sephadex A-50 and Sephadex G-100. Major and minor CA peaksin Sephadex G-100 chromatography showed respective molecularweights of 48,000 and 25,000. The molecular weight of the CAdetermined by polyacrylamide disc gel electrophoresis was 42,000?5,000.The activity of CA was inhibited by ethoxyzolamide (I₅₀=2.8?10^-9M), acetazolamide (I₅₀=2.5?10^-7 M) and sulfanilamide (I₅₀=2.9?10^-6M). (Received January 5, 1984; Accepted April 26, 1984)     

A unique electrophoretic slow-moving glucose 6-phosphate dehydrogenase variant (G6PD Asahikawa) with a markedly acidic pH optimum

Summary A new glucose 6-phosphate dehydrogenase (G6PD) variant associated with chronic nonspherocytic hemolytic anemia was discovered in Japan. The patient showed hemolytic crises after upper respiratory infections. The enzyme activity was about 3.8% of the normal. The partially purified enzyme revealed slow anodal electrophoretic mobility, high Km NADP, marked thermal-instability, and increased affinity for a substrate analogue (deamino-NADP). A particular characteristic of this enzyme was a biphasic pH curve with a greatly increased activity at low pH values. From these results, this variant was clearly different from hitherto observed G6PD variants, and was designated G6PD Asahikawa.     

Evidence that endogenous 6-(ARG or LYS)-opioid peptides can interact with kappa-receptors as agonists

Evidence is provided for the abilities of endogenous 6-(Arg or Lys)-opioid peptides to interact with kappa-receptors as agonists. Dynorphin-(1-17) and -(1-8), alpha- and beta-neo-endorphin, [Met5]-enkephalin-Arg6-Phe7 and des acetyl salmon endorphin I significantly inhibited the electrically-evoked contractions of rabbit vas deferens which had been shown to contain kappa-receptors exclusively, indicating that endogenous 6-(Arg or Lys)-opioid peptides could act on kappa-receptors as agonists. Additionally, the inhibition of contractions of rabbit vas deferens by 6-(Arg or Lys)-opioid peptides was antagonized more effectively by Mr 2266 which had a high affinity to both mu- and kappa-receptors, than naloxone which had a high affinity only to mu-receptors. This also suggested that 6-(Arg or Lys)-opioid peptides acted as kappa-receptor agonists. The rank order of the inhibitory potency of 6-(Arg or Lys)-opioid peptides against contractions of rabbit vas deferens was as follows: dynorphin-(1-17) greater than alpha-neo-endorphin greater than beta-neo-endorphin .=. dynorphin-(1-8) greater than des acetyl salmon endorphin I greater than [Met5]-enkephalin-Arg6-Phe7. Since other endogenous opioid peptides such as [Met5]- and [Leu5]-enkephalin and beta-endorphin have been shown not to act on kappa-receptors as agonist, data in the present study suggest that endogenous opioid peptides can be classified into two groups in terms of an ability to interact with kappa-receptors as an agonist.     

Nitrobacter agilis Cytochrome c-550: Isolation, Physicochemical and Enzymatic Properties, and Primary Structure

Nitrobacter agilis cytochrome c-550 was purified to an electrophoreticallyhomogeneous state, and some of its properties were determined.The cytochrome showed an absorption peak at 410 nm in the oxidizedform, and peaks at 416, 521 and 550 nm in the reduced form.Its isoelectric point was 8.1 at 5?C. Analysis of the aminoacid composition showed that the cytochrome molecule was composedof 108 amino acid residues, 16 of which were lysine residues. The cytochrome reacted rapidly with N. agilis cytochrome c oxidaseand yeast cytochrome c peroxidase and more slowly with Pseudomonasaeruginosa nitrite reductase and bovine cytochrome c oxidase.The reactivities with these redox enzymes suggested that thecytochrome might be an evolutionary stage between bacterialand eukaryotic cytochromes c. The primary structure of the cytochrome from the N-terminusto the 85th residue was determined. The N-terminal sequencewas homologous to the corresponding portion of the primary structureof horse cytochrome c. ¹ Present adress: Department of Chemistry, Faculty of Science,Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo,152, Japan. (Received December 3, 1981; Accepted January 28, 1982)     

Kinetics of DNA replication in the Indian muntjac chromosomes as studied by quantitative autoradiography

DNA replication patterns of individual chromosomes and their various euchromatic and heterochromatic regions were analyzed by means of quantitative autoradiography. The cultured cells of the skin fibroblast of a male Indian muntjac were pulse labeled with ³H-thymidine and chromosome samples were prepared for the next 32 h at 1–2 h intervals. A typical late replication pattern widely observed in heterochromatin was not found in the muntjac chromosomes. The following points make the DNA replication of the muntjac chromosomes characteristics: (1) Heterochromatin replicated its DNA in a shorter period with a higher rate than euchromatin. (2) Two small euchromatic regions adjacent to centromeric heterochromatin behaved differently from other portions of euchromatin, possessing shorter Ts, higher DNA synthetic rates and starting much later and ending earlier their DNA replication. (3) Segmental replication patterns were observed in the chromosomes 2 and 3 during the entire S phase. (4) Both homologues of the chromosome 3 showed a synchronous DNA replication pattern throughout the S phase except in the distal portion of the long arms during the mid-S phase.     

Achromobacter protease I-catalyzed conversion of porcine insulin into human insulin

We have established a procedure for converting porcine insulin into human insulin using a serine protease from $\text{Achromobacter}\text{lyticus}$ M497-1 which shows unique specificity against lysine residues on the carboxyl side of the splitting point. Desalanine-(B30)-insulin (DAI) was prepared by digestion of porcine insulin with $\text{Achromobacter}$ protease. The coupling between DAI and Thr-OBu^t was performed by the same enzyme at pH 6.5 with a large excess of the amine component (Thr-OBu^t) in the presence of high concentrations of organic co-solvents. The highest yield was 85% by 20 h reaction at 37°C. The synthesized [Thr-OBu^t-B30]-insulin was isolated, then deprotected with trifluoroacetic acid in the presence of anisole to obtain semisynthetic human insulin.     

Pathological and microbiological studies on calf pneumonia occurring in mass rearing facilities

Pathological and microbiological studies were conducted on lesions in the lungs of 194 calves from mass rearing facilities. Macroscopically, the lesions were classified into six forms: nonlesion, atelectasis, mild pneumonia, moderate pneumonia, advanced pneumonia, and advanced pneumonia accompanied with abscess. Histopathological examination revealed bronchopneumonia in most of the calves. Lesions more advanced than moderate pneumonia were complicated with desquamation, severe exudation, and necrosis. Bacteriologically, Pasteurella sp. was isolated often in combination with Staphylococcus sp. from about a half of the atelectatic cases. With the development of pneumonic lesions, Pasteurella sp. was isolated at a high frequency in combination with Haemophilus sp., Streptococcus sp., and Corynebacterium sp. Prominent necrosis was more often seen in cases with Pasteurella haemolytica isolated than in cases with only Pasteurella multocida isolated. Mycoplasma sp. and Ureaplasma sp. were isolated from distinctly pneumonic lesions. Advanced pneumonic lesions were observed in many calves over 30 days of age. The importance of environmental and managerial improvement was also emphasized, since calf pneumonia tended to break out in facilities under unsatisfactory conditions in the present work.     

Giant oil cells in the cotyledons ofPharbitis nil and other convolvulacean plants

Suspension of protoplasts (ca. 13–25 μm in diameter) that were isolated from the mesophyll of the cotyledons ofPharbitis nil, strain Violet, contained many large spherical or spheroidal bodies (ca. 100 μm in diameter). Microscopic observation of these bodies and some anatomic studies of the cotyledons during embryogenesis and after germination showed that these bodies are giant cells containing many oil drops stainable with Sudan dyes. Such giant cells were found in four otherPharbitis nil strains, Nepal, Tendan, Africa and Tokyo-kokei, and in six other Convolvulacean plants,Ipomoea batatas, cv. Koukei-14,Calystegia japonica, Calystegia hederacea, Calonyction aculeatum, Quamoclit pennata andCuscuta japonica.     

Concentration-dependent differntial effects of cortisol on synthesis of α-lactalbumin and of casein in cultured mouse mammary gland explants: Importance of prolactin concentration

Summary Cortisol was previously shown to elicit a concentration-dependent inhibition of α-lactalbumin accumulation in midpregnant mouse mammary gland cultured in medium containing optimal concentrations of 5 μg/ml prolactin and insulin. In contrast, casein accumulation under these conditions was progressively stimulated by addition of increasing amounts of cortisol (Ono, M.; Oka, T. Cell 19: 473–480; 1980). In the present study we found that in the presence of a suboptimal concentration of 0.5 μg/ml prolactin, 2.8×10⁻⁹ M to 2.8×10⁻⁷ M cortisol stimulated α-lactalbumin accumulation. Furthermore, higher concentrations of cortisol produced a smaller inhibition of α-lactalbumin accumulation as compared to that obtained in cultures containing 5 μg/ml prolactin. The maximal increase in α-lactalbumin accumulation attained in the presence of 1.4×10⁻⁸ M cortisol, 0.5 μg/ml prolactin, and insulin was comparable to that observed in culture containing 5 μg/ml prolactin and insulin. Similar results were obtained in a cortisol concentration-response study of α-lactalbumin accumulation in cultures containing a suboptimal concentration of 0.5 μg/ml human placental lactogen. Measurement of the rate of α-lactalbumin synthesis in cultured tissue indicated that the opposing effects of low and high concentrations of cortisol on α-lactalbumin accumulation involved an alteration in the rate of synthesis of the milk protein. In contrast to α-lactalbumin, the synthesis of casein was stimulated in a concentration-dependent manner by addition of cortisol that acted synergistically with either 0.5 μg/ml or 5 μg/ml prolactin. The maximal increases were obtained in the presence of 2.8×10⁻⁶ M cortisol. These results indicated that the action of cortisol on α-lactalbumin accumulation can be modulated by the concentration, of prolactin and suggest that the interplay between cortisol and prolactin in regulation of α-lactalbumin synthesis may be different from that involved in casein synthesis.