High-density Escherichia coli cultivation process for hyperexpression of recombinant porcine growth hormone.

Fermentation studies were performed on an Escherichia coli culture that carries a recombinant plasmid composed of an ampicillin-resistant gene, a temperature-regulated pL promoter, and a porcine pituitary cDNA sequence coding for growth hormone. The objective was to achieve high cell density while maintaining the specific expression level of recombinant porcine growth hormone (r-pGH) observed in shake flasks. At a specific expression level of 20% of total cell protein, the cell density of a glucose-limited fed-batch process reached 38 units of OD600 in 14 h, compared to flask cultivation, which resulted in only 1.4 units of OD600 in the same period. The observed critical fermentation conditions for maximal expression included (1) limiting glucose concentration below 1 g l-1 throughout the fed-batch growth and induction phases, (2) keeping postinduction temperature at 42 degrees C for 5-7 h, and (3) maintaining a postinduction growth rate around 0.17-0.21 h-1.     

Release of different fractions of epidermal growth factor from human platelets in vitro: preferential release of 140 kDa fraction.

Platelet-rich plasma in acidic-citrate-dextrose anticoagulant was kept for 5 days in an oxygen-permeable bag at 22 degrees C in an incubator/rotator. Platelet count remained stable throughout the experiment. On days 0, 3 and 5, aliquots were removed; platelets were isolated by centrifugation at 22 degrees C, 1500 g for 20 min, reconstituted to the original volume with PBS buffer, and the contents of alpha-granules were released by repeated freezing and thawing. Epidermal growth factor (EGF) and beta-thromboglobulin (beta-TG) in the platelet-poor plasma and platelet lysates were determined by radioimmunoassays. Results indicated that in platelet-free plasma, both total EGF and beta-TG increased 3-5-fold after 5 days; this amount represented 10-20% of the factors stored in the platelets. Correspondingly, the EGF and beta-TG contents of the platelet lysates exhibited accompanying decreases. HPLC fractionation showed that the main EGF fraction which progressively decreased in the lysates and increased in plasma had a molecular mass of 140 kDa. The contents of the 67 kDa and 6 kDa fractions did not change substantially. We conclude that under these conditions, the 140 kDa fraction was released preferentially. In view of these and previous experiments, it seems likely that different organs contribute to plasma EGF fractions.     

Influx of extracellular calcium is required for the membrane translocation of 5-lipoxygenase and leukotriene synthesis.

Our studies assessed the effects of increases in intracellular calcium concentrations [( Ca2+]i) on leukotriene synthesis and membrane translocation of 5-lipoxygenase (5LO). The calcium ionophore ionomycin and the tumor promoter thapsigargin stimulated leukotriene production and translocation of 5-lipoxygenase to the membrane. Both agents elicited prolonged rises in [Ca2+]i. Leukotriene C4 production associated with [Ca2+]i in cells stimulated with various concentrations of ionomycin and thapsigargin suggests that a threshold [Ca2+]i level of approximately 300-400 nM is required. In the absence of extracellular Ca2+, both the ionomycin- and thapsigargin-induced rises in [Ca2+]i were transient, indicating that the prolonged [Ca2+]i elevation is due to an influx of extracellular Ca2+. Addition of EGTA to the external medium before, or at different times during, the treatment with ionomycin or thapsigargin instantaneously inhibited 5LO translocation and leukotriene synthesis, indicating that Ca2+ influx plays an essential role in 5LO membrane translocation and leukotriene synthesis. No leukotriene production was detected when cells were stimulated by a physiological stimulus of leukotriene D4. The addition of 100 nM leukotriene D4 triggered peak rises in [Ca2+]i that were comparable to those achieved by the ionomycin and thapsigargin. However, the leukotriene D4 induced rise was transient and rapidly declined to a lower but still elevated steady-state level, which was attributed to Ca2+ influx. Stimulation with 100 nM leukotriene D4 for 15 s increased the cellular levels of 1,4,5-inositol triphosphate (IP3), 1,3,4-IP3, and 1,3,4,5-inositol tetraphosphate (IP4).(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbohydrate, Amino acid, Phenolic and Mineral Nutrient Contents of Pepper Plants in Relation to Age-Related Resistance to Phytophthora capsici

The relationship between age-related resistance of peper plants to Phytophthora capsici and contents of carbohydrates, amino acids, phenolics and mineral nutrients in pepper stems was studied using two pepper cultivars, Hanbyul (susceptible) and Kingkun (resistant). With increasing age of pepper plants, the two cultivars, which differ in their susceptibility to Phytophthora blight, became gradually resistant to the disease. The cultivar Kingkun distinctly showed the age-related resistance to Phytophthora blight at the second branch stage. The weight of dry matter in healthy stems of pepper plants at the second branch stage was twice that at the six leaf stage. The resistant cultivar Kingkun contained lower levels of fructose, glucose and sucrose in stems than the susceptible cultivar Hanbyul at the different developmental stages. No consistent differences between the developmental stages of the plants were recognized with regard to their glucose content. However, the contents of fructose and sucrose in the cultivar Hanbyul greatly increased at the second branch stage. The levels of inositol reduced in both pepper cultivars during plant development. In view of the fact that there were only slight changes in the amount of total amino acids, it seems unlikely that there is a relationship between the amino acid metabolism and the retardation of Phytophthora infection during plant development. The amounts of total phenolic compounds in pepper stems were relatively low at the later growth stages of the plants and also in the resistant cultivar Kingkun. The contents of macroelemental nutrients such as nitrogen, phosphorus, potassium, calcium and magnesium were drastically reduced in pepper stems at the later plant growth stage. No significant differences between the cultivars or the plant growth stages were found in the silicon and microelemental nutrients such as sodium, iron, zinc and manganese. These results suggest that the expression of age-related resistance of pepper plants may be due to the morphological and nutritional changes in tissues of pepper stems during ageing, i.e. the pronounced increase in weight of dry matter, the significant decrease in amounts of mineral nutrients such as nitrogen, phosphorus, potassium, calcium and magnesium, and the tow contents of fructose, glucose and sucrose in the stem tissues.     

Processing of Ada protein by two serine endoproteases Do and So from Escherichia coli

Two soluble serine proteases Do and So from Escherichia coli were found to distinctively cleave the purified, 39 kDa Ada protein into fragments with sizes of 12-31 kDa. Protease So appears to generate a C-terminal 19 kDa polypeptide, similarly to OmpT protease. In addition, the purified 19 kDa C-terminal half of Ada protein can be further processed mainly to an 18 kDa fragment by protease So and to a 12 kDa by protease Do. These results suggest that proteases Do and So are involved in endogenous cleavage of Ada protein, which may play a role in down-regulating the adaptive response to alkylating agents.     

Proton transport by bacteriorhodopsin in planar membranes assembled from air-water interface films

Bacteriorhodopsin, in known amounts and controlled orientation, is incorporated into planar membrane films. These films are formed by the sequential transfer of two air-water interface films onto a thin, hydrophilic, electrically conductive support cast from nitrocellulose. The films are easily accessible to electrical measurements and to control of the ionic milieu on either side of the membrane. The area of the assembled membrane films can be varied between 2.3 x 10(-2) cm2 and 0.7 cm2. Illumination of these films produces photocurrents, photovoltages, and changes in the pH of the surrounding medium. The peak amplitude of the photocurrent increases linearly with light intensity for dim lights, and it approaches a saturating value for brighter lights. In the linear range, the stoichiometry of transport is 0.65 +/- 0.06 protons/absorbed photon. The rate of transport is linearly proportional to light at all intensities tested. The amplitude and kinetics of the photovoltage measured are accurately predicted by the photocurrent generated and the passive electrical features of the film. Parallel measurements of pH and photocurrent reveal that the light-induced changes in pH are fully accounted for by the rate and amount of charge transport across the membrane. Preceding the transport of protons, a transient photovoltage is detected that exhibits no detectable latency, reaches peak in about 80 microseconds, and probably arises from light-induced intramolecular charge displacements.     

Age as a factor in 6-hydroxydopamine-induced plasticity in the hypothalamus

Summary The question of age as a possible factor influencing the regenerative response of catecholaminergic varicosities in the hypothalamus was investigated in the supraoptic commissure and the paraventricular, periventricular, and dorsomedial hypothalamic nuclei of rats that had received intraventricular injections of the neurotoxin 6-hydroxydopamine when they were (1) neonates, (2) young adults, or (3) senescent adults. After postneurotoxin survival for 4, 21, 56, or 180 days, the animals were perfused, and the hypothalamic tissue sections were cut and processed using a glyoxylic acid method for localizing catecholamines. Four days following neurotoxin administration, counts of fluorescent varicosities showed a significant loss of catecholamine varicosities in each of the four areas. Subsequently, at least partial restoration of numbers of catecholamine varicosities occurred in all hypothalamic areas in all three age groups. It is concluded that, following selective lesions induced by the neurotoxin 6-OH-DA, catecholamine varicosities were restored both in immature and mature groups. According to the evidence obtained experimentally, the rate of restoration was greater in the neonate group, whereas the percentage restoration attained varies according to the hypothalamic area studied and the age of the animal.     

Interference of ATP and acidity in the determination of inorganic phosphate by the Fiske and Subbarow method.

The influences of ATP and trichloroacetic acid on the color development of molybdenum blue in the Fiske and Subbarow method for the determination of inorganic phosphate were investigated. From these results optimum concentrations of ATP and trichloroacetic acid were recommended. Satisfactory results were obtained in the determination of the ATPase activity of sarcoplasmic reticulum membranes isolated from rabbit skeletal muscle.     

Isolation and Genetic Analysis of Mutant Strains of CHLAMYDOMONAS REINHARDI Defective in Gametic Differentiation

Impotent mutant strains of Chlamydomonas reinhardi, mating-type (mt) plus, are described that have normal growth and motility but fail to differentiate into normal gametes. Procedures for their isolation and their genetic analysis are described. Five of the imp strains (imp-2, imp-5, imp-l, imp-7, and imp-8) exhibit no flagellar agglutination when mixed with mt- or mt+ gametes and the mutations are shown to be unlinked to the mt locus (with the possible exception of imp-7). Two of the strains (imp-3 and imp-4) carry leaky mutations that affect cell fusion; neither mutation is found by tetrad analysis to be linked to mt or to the other. Cells of the imp-1 strain agglutinate well with mt- gametes and active agglutination continues for up to 48 hours, but cell fusion occurs only very rarely. Analysis of these rare zygotes indicates that imp-1 is closely linked to the mt+ locus, and fine-structural studies reveal that imp-1 gametes produce a mutant mating structure involved in zygotic cell fusion. The development of sexuality in C. reinhardi therefore appears amenable to genetic dissection.