Over-production of Thermus protease in dense culture of Escherichia coli using two carbon sources

Escherichia coli JM109(DE3) harboring expression plasmid pkAQNÆC30, which carries the Thermus protease aqualysin I (AQI) gene, was cultivated with glucose as a sole carbon source. The final cell concentration was over 15 g dry weight/l and the amount of AQI produced reached approximately 130 kU/ml broth. Moreover, by using two carbon sources, glucose and glycerol, the production yield was increased to over 200 kU AQI/ml, while suppressing the formation of inhibitory acetic acid.     

Mouse {beta}-galactoside {alpha}2,3-sialyltransferases: comparison of in vitro substrate specificities and tissue specific expression

Four types of ß-galactoside     

Optimal strategy of plant antiherbivore defense: Implications for apparency and resource-availability theories

Plants produce chemicals as methods against animal herbivory. Such chemical defenses are classified into two major categories: (i) quantitative defenses with massive production of indigestible substances; and (ii) qualitative defenses with production of poisonous substances. A mathematical model was developed that identified factors that favored the evolution of quantitative defenses. Selecting an annual plant for simplicity, the allocation of photosynthetic production between growth substances and defense substances was considered. If the plant invests more in defense substances, it can protect itself more efficiently from herbivory but with a reduced growth rate. If it invests more in growth substances, the contrary holds. Using Pontoryagin's maximum principle, the following results were obtained: (i) the plant should conduct quantitative defenses when the growth rate (G), reflecting resource-availability, is low and the growth period (T) is long as well; (ii) if the plant invests in quantitative defenses, the optimal proportion of defense substances (χ^*) should be higher asG is smaller, but it is independent ofT; and (iii) the value of χ^* is not monotone for the effectiveness of defense substance (A), but has a maximum at an intermediate value ofA. Predictions of the model partly supported both Feeny's apparency theory, claiming that apparent plants or their parts for herbivores should quantitatively defend themselves, and Coley's resource-availability theory, claiming that plants with rich resources should invest in growth rather than defense.     

Electron-microscope cytochemistry of acetylcholine-like cation by means of low-temperature "ionic fixation"

A fresh preparation of frog neuromuscle was fixed at low temperatures (0 degree-4 degrees C) by means of an "ionic-fixation" procedure which is based on the precipitation of quaternary ammonium cations, such as choline and acetylcholine, with molybdic or tungstic heteropolyanions. A low temperature was used to slow down drastically the biological processus of vesicular exocytosis so that ionic fixation, the speed of which is only slightly influenced by temperature variation, could be performed efficiently. In addition to the conventional point-like precipitate in the synaptic vesicle which is considered to be vesicular acetylcholine, numerous spot-like precipitates were observed in the synaptic cleft. Most of these were contiguous to the active zone, and some were in a paired form and corresponded to the double rows of the synaptic vesicles in contact with active zones. It is concluded that these spot-like precipitates were acetylcholine-like cations of the synaptic vesicles which had been discharged into the synaptic cleft by exocytosis and captured by the ionic fixation procedure. The results are discussed in relation to the vesicular and non-vesicular hypothesis of acetylcholine release.     

Separation and properties of five glycosaminoglycan sulfatases from rat skin.

Chondroitin sulfates, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, and oligosaccharides derived from these sulfated glycosaminoglycans have been used for the measurement of sulfatase activity of rat skin extracts. Chromatographic fractionation of the extracts followed by specificity studies demonstrated the existence of five different sulfatases, specific for 1) the nonreducing N-acetylglucosamine 6-sulfate end groups of heparin sulfate and keratan sulfate, 2) the nonreducing N-acetylgalactosamine (or galactose) 6-sulfate end groups of chondroitin sulfate (or keratan sulfate), 3) the nonreducing N-acetylgalactosamine 4-sulfate end groups of chondroitin sulfate and dermatan sulfate, 4) certain suitably located glucosamine N-sulfate groups of heparin and heparan sulfate, or 5) certain suitably located iduronate sulfate groups of heparan sulfate and dermatan sulfate. Two arylsulfatases, one of which was identical in its chromatographic behaviors with the third enzyme described above, were also demonstrated in the extracts. These results taken together with those previously obtained from studies on human fibroblast cultures suggest that normal skin fibroblasts contain at least five specific sulfatases and diminished activity of any one may result in a specific storage disease.     

Myocardial adaptations in aortic valve disease.

Injections of human chorionic gonadotropin (HCG) have been claimed to aid in weight reduction by reducing hunger, and affecting mood as well as aiding in localized (spot) reduction. We have tested these claims in a double-blind randomized trial using injections of HCG or placebo. Weight loss was identical between the two groups, and there was no evidence for differential effects on hunger, mood or localized body measurements. Placebo injections, therefore, appear to be as effective as HCG in the treatment of obesity.     

Requirement of the core structure of a complex-type glycopeptide for the binding to immobilized lentil- and pea-lectins

Structural requirements for the binding of oligosaccharides and glycopeptides to immobilized lentil- and pea-lectins were investigated by use of radioactively-labeled glycopeptides and oligosaccharides. The results indicate that an intact 2- acetamido-2-deoxy-β-d-glucopyranosyl residue at the reducing end of a complex-type oligosaccharide is essential for high-affinity binding to lentil lectin-Sepharose but not to concanavalin A-Sepharose and that an asparagine residue is required for the binding of a complex-type glycopeptide to pea lectin-Sepharose. In addition, interaction of a complex-type oligosaccharide with lentil lectin-Sepharose was enhanced by exposure of nonreducing, terminal 2-acetamido-2-deoxy-β-d-glucopyranosyl groups, whereas interaction with pea lectin-Sepharose was enhanced only after exposure of nonreducing, terminal α-d-mannopyranosyl groups.     

Development of the Formula-Detoxification System during Adaptation to Air of Submerged Rice Seedlings

Changes in activities of enzymes and levels of antioxidant substratesinvolved in the -detoxification system in seedlings of rice (Oryza sativa L. cv. Yamabiko) inresponse to variations in the oxygen environment were studied.Activities of superoxide dismutase, ascorbate peroxidase, monodehydroascorbatereductase, dehydroascorbate reductase, glutathione reductaseand catalase, expressed either on the basis of fresh weightof shoots or relative to levels of soluble protein were muchlower in seedlings germinated under water for 6 days than inthose germinated in air for the same period of time. When submergedseedlings were exposed to air, the activities of these six enzymesincreased to or exceeded the levels in aerobically grown controlsduring 24 h of adaptation to air. Ascorbate and glutathione,which act as antioxidant substrates in the -detoxification system, were present in submergedseedlings at nearly the same levels as those found in aerobicallygrown controls. On exposure of submerged seedlings to air, thelevel of ascorbate in creased slightly, but the level of glutathioneshowed a rapid increase, reaching 7 times that in aerobicallygrown controls within 12 h of adaptation to air. Levels of allsix antioxidative enzymes and of two substrates involved inthe detoxification of the superoxide radical increased withincreases in oxygen tension in the environment. Moreover, thedevelopment of this system consisted of two steps, namely, arapid increase in the level of glutathione and a subsequentslow increase in the activities of antioxidative enzymes. ¹ Present address: Research Institute for Bioresources, OkayamaUniversity, Kurashiki, 710 Japan.     

Human placental sialidase complex: characterization of the 60 kDa protein that cross-reacts with anti-saposin antibodies

Sialidase isolated from human placenta is associated with several proteins including acid beta-galactosidase, carboxypeptidase, N-acetyl-alpha-galactosaminidase, and others. These proteins are thought to form an aggregated complex during isolation of sialidase. One of the proteins of 60 kDa was recently identified by Potier et al. (Biochem. Biophys. Res. Comm. 173, 449-456, 1990) as a sialidase protein: this protein also cross-reacted with anti-prosaposin antibodies. We have isolated this protein and from the following evidence identified it as a heavy chain component of immunoglobulin G and not sialidase or a derivative of prosaposin. On gel filtration HPLC, sialidase activity and the 60 kDa protein were clearly separated from one another. The 60 kDa protein cross-reacted not only with antibodies raised against human saposins A, C, and D, but also with second antibody (goat anti-rabbit immunoglobulin G antibody) alone. This 60 kDa protein strongly cross-reacted with anti-human immunoglobulin G antibodies. The sequence of the initial 15 amino acids from the N-terminus of the 60 kDa protein was identical to the sequence of an immunoglobulin G heavy chain protein Tie (gamma 1).