Purification and characterization of pig lung carbonyl reductase.

A pyrazole-sensitive carbonyl reductase from pig lung was purified to homogeneity by electrophoretic criteria. Chemical cross-linking study suggested that the native enzyme is a tetramer with a Mr of 103,000, consisting of apparent identical subunits of Mr 24,000. The enzyme reduced aliphatic and aromatic carbonyl compounds with NADPH as a preferable cofactor to NADH and catalyzed the oxidation of secondary alcohols and the aldehyde dismutation in the presence of NAD(P)+. Immunohistochemical study with the antibodies against the enzyme revealed that the enzyme was localized in the ciliated cells, nonciliated bronchiolar cells, Type II alveolar pneumocytes, and the epithelial cells of the ducts of the bronchial glands in the pig lung. In addition to the properties and distribution, the pig lung enzyme was immunochemically similar to the pulmonary enzymes in the guinea pig and mouse. However, the pig enzyme showed the following unusual features. (1) The enzyme exhibited an equatorial specificity in the reduction of 3-ketosteroids; the 4-pro-S hydrogen of NADPH was transferred to the carbonyl carbon atom of 5 alpha- and 5 beta-androstanes, and the respective reduced products were identified as 3 beta- and 3 alpha-hydroxysteroids. (2) Although the NADPH-linked reduction of carbonyl compounds apparently obeyed the Michaelis-Menten kinetics at pH 6.0, the double-reciprocal plots of the velocity vs concentrations of the carbonyl substrates were convex at pH higher than 6.5. The Hill coefficients and [S]0.5 values for the substrates decreased as the pH for reaction increased. The results suggest that the pig enzyme exhibits negative cooperativity with respect to the carbonyl substrates and that the hydrogen ion acts as an allosteric effector abolishing the negative interaction.     

A Parvalbumin-Like Calcium-Binding Protein Is Present in Plant Leaves

A protein with relatively high homology in its N-terminal aminoacid sequence to animal parvalbumin and oncomodulin has beenidentified in leaves of rice (Oryza sativa L.). The PV-likeprotein has a relative molecular mass of 27,000 and an isoelectricpoint of 5.0. This protein was partially purified by ion-exchangechromatography, and the purified protein was found to have Ca²⁺-bindingactivity in a microscale Ca²⁺-binding assay. Furthermore, anantiserum raised against a synthetic oligopeptide based on theN-terminal amino acid sequence of this protein cross-reactedwith protein not only from rice but also from other monocotyledonousplants. (Received October 17, 1990; Accepted October 17, 1991)     

An encounter with extraterrestrial intelligence]

It is much easier to find extraterrestrial intelligence than to detect simple organisms living on other planets. However, it is hard to communicate with such intelligence without the mutual understanding of inter-stellar communication protocol. The radio SETI (The Search for Extra-Terrestrial Intelligence) was initiated with the pioneering work of F. Drake in 1960, one year after the historical SETI paper by Cocconi and Morrison. This talk explains that SETI evolves with two bases of science; the understanding of our universe and the development of technology. Since SETI has had strong connection with radio astronomy from its early beginning, the impacts of radio astronomical findings and technological breakthrough can be seen in many aspects of the SETI history. Topics of this talk include the detection of microwave 3 K background radiation in the universe. Interstellar atomic and molecular lines found in radio-wave spectra provide the evidence of pre-biotic chemical evolution in such region. Radio telescope imaging and spectral technique are closely associated with methodology of SETI. Topics of the talk extend to new Allen Telescope Array and projected Square Kilometer Array. Recent optical SETI and the discoveries of extra solar planets are also explained. In the end, the recent understanding of our universe is briefly introduced in terms of matter, dark matter and dark energy. Even our understanding of the universe has been evolutionarily revolved and accumulated after 1960, we must recognize that our universe is still poorly understood and that astronomy and SETI are required to proceed hand in hand.     

Parasitic specialization ofGeotrichum candidum citrus race

Parasitic specialization ofGeotrichum candidum citrus race, the causal agent of citrus sour rot, was investigated. Of seven isolates tested for pathogenecity, all could infect ten species of citrus fruits and edible parts of five species of noncitrus crops. Only one isolate (Ap2), isolated from soil of an apple orchard, could infect apple fruit.     

Genetic diagnosis of cytoplasmic male sterile cybrid plants of rice

Twelve Japanese rice cultivars were converted to CMS by asymmetric protoplast fusion with MTC-5A, the cytoplasm of which was derived from an indica rice, Chinsurah Boro II. With the exception of the cybrids that had a nucleus from Hoshiyutaka, most of these cybrid plants were sterile. The unique sequence downstream from the mitochondrial atp6 of MTC-5A was specifically amplified in the sterile cybrid plants by PCR. All progenies of the cybrid plants carrying this unique sequence were sterile. On the other hand, in some of the sterile cybrid plants in which the unique sequence was not amplified by PCR, fertility was recovered in their progenies. Somaclonal mutation may have caused sterility in these cybrids. Only the cybrid plants that had the unique sequence detected by PCR were CMS. Thus, the CMS plants can be selected rapidly and easily by PCR, at an early stage of plant regeneration. Soon after transplanting the regenerated plants to a green house, fertile cybrids and sterile cybrids produced by somaclonal mutation can be removed. These findings also show that the unique region downstream from atp6 is tightly linked with the CMS phenotype.     

Regulation of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activation by Inorganic Phosphate through Stimulating the Binding of the Activator CO2 to the Activation Sites

The mechanism of the regulation of the activation of ribulose1,5-bisphosphate carboxylase/ oxygenase (RuBisCO) by inorganicphosphate (P_i) in the presence of limiting concentrations ofCO₂ was explored. The activation state of RuBisCO increasedsigmoidally following a biphasic kinetics against the concentrationof P_i in the activation mixture with an intermediary plateauat 2 to 3 mM P_i when the enzyme was activated for 30 min. Theintermediary plateau could not be seen when the preincubationtime was 10 min and the activation was completed at 10 mM P_i.RuBisCO from Euglena also showed a quite similar activationkinetics. The activation was not due to the contaminating CO₂included in the stock P_i solution or in the activation buffercontaining the enzyme. The experiments with 2-carboxyarabinitol1,5-bisphosphate showed that the P_i stimulated activation wasdue to the promotion of binding of the activator CO₂ to theactivation sites. It was also found that P_i increased the affinityof RuBisCO for the activator CO₂ 5.4-fold accompanied by a decreaseof the half-saturating concentration of CO₂ to 1.6 µMat 20 mM MgCl₂. Physiological significance of the effects ofP_i on the activation of RuBisCO is discussed. ²Present address: Laboratory of Plant Molecular Physiology,Research Institute of Innovative Technology for the Earth (RITE),9-2 Kizugawadai, Kizu-cho, Soraku-gun, Kyoto, Japan.     

Efficient production of anti-(hepatitis B virus) antibodies and their neutralizing activity in chimpanzees

 For industrial production of human monoclonal antibodies (hmAb) against hepatitis B virus surface antigen (HBsAg), we scaled-up a short-term perfusion culture in serum-free medium, which was chosen as the most suitable culture method, to a 50-l fermentor equipped with a rotating shear filter. Using hydrophobic chromatography as the initial step of hmAb purification, the mAb HBW4, HBW6 and W471 were isolated in good quality from the respective culture broths in yields of approximately 75%. Each of the three purified hmAb alone, and a cocktail of the three, protected chimpanzees against HB virus, when injected intravenously 3 h after viral challenge, as long as the serum antibody levels were significant. A pharmacokinetic study using cynomolgus monkeys demonstrated that the hmAb have a long plasma half-life and bioavailability of approximately 76% upon intramuscular injection in primates. Thus, anti-HBsAg hmAb produced by an industrial process are expected to be successfully used in clinical fields. Received: 20 June 1994/Received revision: 16 September 1994/Accepted: 10 October 1994     

Effects of fungal pretreatment and steam explosion pretreatment on enzymatic saccharification of plant biomass

The effects of consecutive treatments by a lignin-degrading fungus Phanerochaete chrysosporium and by steam explosion for the enzymatic saccharification of plant biomass were studied experimentally, and the optimal operational conditions for obtaining the maximum saccharification were evaluated. Beech wood-meal was treated by the fungus for 98 days and then by high steam temperatures of 170-230 degrees C with steaming times of 0-10 min. The treatment of the wood-meal by fungus prior to steam explosion enhanced the saccharification of wood-meal. The treated wood-meal was separated into holo-cellulose, water soluble material, methanol soluble lignin, and Klason lignin. The saccharification decreased linearly with the increase in the amount of Klason lignin. It was estimated by the equation for the saccharification of exploded wood-meal expressed as a function of steam temperature and steaming time that the maximum saccharification of wood-meal was obtained by consecutive treatments such as fungal treatment for 28 days and then steam explosion at a steam temperature of 215 degrees C and a steaming time of 6.5 min. (c) 1995 John Wiley & Sons, Inc.     

Changes in cytokeratin expression in epidermal keratinocytes during wound healing

In order to investigate the re-epithelialization process during wound healing, the hair on the back of guinea pigs was shaved and then excisional wounds were made through the entire thickness of the skin. Histological changes were observed and changes in the expression of different cytokeratin polypeptides were examined using an immunohistochemical technique. Immunohisto chemical study revealed that the proliferating and migrating keratinocytes expressed the same cytokeratins as the basal cells of normal epidermis. In addition, the entire epidermis of fairly remote areas from the edges of the wound where no thickening was observed showed a temporarily abnormal staining pattern. The suprabasal cells in the regenerating epidermis temporarily expressed cytokeratins not only specific for suprabasal cells but also specific for basal cells. The cytokeratins expressed in normal basal keratinocytes were also present in the thickened granular layers. These data indicate that the expression of cytokeratins in the epidermal keratinocytes (even in fairly remote areas from the wound edges) changes during wound healing, that the origin of the migrating keratinocytes from the remaining epidermis seems to be the basal cells in the epidermis, and that the appearance of keratohyalin granules is not related to changes in cytokeratin expression.     

Plasmodium falciparum protein associated with the invasion junction contains a conserved oxidoreductase domain

The merozoite cap protein-1 (MCP-1) of Plasmodium falciparum follows the distribution of the moving Junction during invasion of erythrocytes. We have cloned the gene encoding this protein from a cDNA library using a monoclonal antibody. The protein lacks a signal sequence and has no predicted trans-membrane domains; none of the antisera reacts with the surfaces of intact merozoites, indicating that the cap distribution is submembranous. MCP-1 is divided into three domains. The N-terminal domain includes a 52-amino-acid region that is highly conserved in a large family of bacterial and eukaryotic proteins. Based on the known functions of two proteins of this family and the pattern of amino acid conservation, it is predicted that this domain may possess oxido-reductase activity, since the active cysteine residue of this domain is invariant in all proteins of the family. The other two domains of MCP-1 are not found in any other members of this protein family and may reflect the specific function of MCP-1 in invasion. The middle domain is negatively charged and enriched in glutamate; the C-terminal domain is positively charged and enriched in lysine. By virtue of its positive charge, the C-terminal domain resembles domains in some cytoskeleton-associated proteins and may mediate the interaction of MCP-1 with cytoskeleton in Plasmodium.