Characterization of immune type interferon (IFN gamma)-induced cytoplasmic protein kinase activity in mouse L cells

Mouse immune interferon (IFNγ) induced double-stranded RNA-independent protein kinase activity in the cytoplasmic fraction of mouse L cells as measured against a histone substrate. Chromatographic purification separated the activity into three distinct kinases of molecular weights of approximately 100K (kinase I), 70K (kinase II), and 70K (kinase III). Partially purified IFNγ was as effective as crude in inducing protein kinase activity. Induction was blocked by treatment of IFNγ with specific anti-IFNγ antibody or by treatment of the L cells with actinomycin D. Kinase II activity was different from that of kinases I and III in that it showed Ca-dependence in the absence of Mg²⁺, was inhibited in activity by the SH binding agent N-ethylmaleimide, and could use the cellular enzymes RNase A and hexokinase as substrates. The described protein kinases could play an important role in mediation of IFNγ effects, particularly where phosphorylated enzyme substrates were shown to have altered activity.     

Fibroblastlike primary cells from human colon adenocarcinoma explants: Collagen biosynthesis

Summary Fibroblastlike primary cells have been obtained from human colon adenocarcinoma explants. Such cells disappear during cell culture and thus have not been previously studied. These cells have a number of altered phenotypic characteristics: a) morphology; b) growth behavior and adherence to culture substrate (they required 3 h for 90% attachment and only presented a flattened morphology 40 h after platting); and c) collagen metabolism. Increased protein biosynthesis (about double than control colon-derived fibroblasts) and maintained ability for collagen biosynthesis have been observed for the tumor-associated fibroblastlike cells. Thus, the collagen to noncollagenous proteins ratio was decreased for these cells. They exhibited an altered type I:type III collagen (5:1 instead of 3:1 in colon fibroblasts) and procollagen (2:1 against 5:1 in colon fibroblasts) ratios as well as a decreased secretion of collagen with an abnormal deposition of procollagens in the cell layer. These studies show a permanent phenotypic alteration in the tumor-associated fibroblastlike cells.     

Detection of salmonellas by DNA hybridization with a fluorescent alkaline phosphatase substrate.

This study evaluates a DNA hybridization assay for salmonella with AttoPhos (JBL Scientific, San Luis Obispo, CA), a fluorescent substrate for alkaline phosphatase. The probe used (50 ng/ml) was a biotinylated 600 bp fragment consisting of a tandem repeat of an insertion sequence (IS200) found in most Salmonella spp. evaluated. The hybridization was carried out at 65 degrees C for 2 h without prior prehybridization and hybrids were detected by the addition of a streptavidin-alkaline phosphatase conjugate. Circles (5 mm) were cut from the membrane and placed in a cuvette containing 1 ml of 1 mmol/l AttoPhos. The reaction was evaluated after 30 min at 37 degrees C with a fluorometer with an excitation wavelength of 440 nm and an emission wavelength of 550 nm. The sensitivity of the probe was estimated to be 10,000 copies of target DNA or 5 x 10(-20) mol of DNA. All 74 salmonella strains tested reacted with the probe but none of the 98 heterologous species tested gave positive results. The results of this study indicate that our assay method, which employs a biotinylated tandem repeat of IS200 and AttoPhos, is a specific and highly sensitive quantitative method for the detection of salmonellas.     

Application of empirical design methodologies to the study of the influence of reaction conditions and N-alpha-protecting group structure on the enzymatic X-Phe-Leu-NH(2) dipeptide synthesis in buffer/dimethylformamide solvents systems

The influence of five different N-terminal protecting groups (For, Ac, Boc, Z, and Fmoc) and reaction conditions (temperature and dimethylformamide content) on the alpha-chymotrypsin-catalyzed synthesis of the dipeptide derivative X-Phe-Leu-NH(2) was studied. Groups such as For, Ac, Boc, and Z always rendered good peptide yields (82% to 85%) at low reaction temperatures and DMF concentrations, which depended on the N-alpha protection choice. Boc and Z were the most reactive N-alpha groups and, in addition, the most suitable for peptide synthesis. On the other hand, the use of empirical design methodologies allowed, with minimal experimentation and by multiple regression, to deduce an equation, which correlates the logarithm of the first order kinetic constant (log k') with reaction temperature, DMF concentration, and hydrophobicity (log P values) of the different protecting groups. The predictive value of the equation was tested by comparing the performance of another protective group, such as Aloc, with the performance predicted by said equation. Experimental and calculated k' values were found to be in good agreement.     

Transient accumulation and subsequent rapid loss of messenger RNA encoding high molecular mass CD45 isoforms after T cell activation.

High molecular mass isoforms of CD45 protein tyrosine phosphatase, predominantly CD45RA, are expressed on naive T cells with increasing density during the first 2 days of cellular activation, and subsequently down-regulated concurrent with increasing expression of the low Mr isoform, CD45RO. We show this to be de novo synthesis of CD45RA dependent upon both RNA and protein synthesis. Using a probe shown to detect mRNA encoding the alternatively spliced abc, ab, bc, and b exon isoforms of CD45, the expression of CD45 was analyzed. Kinetic studies of the transition from high to low Mr CD45 mRNA indicate an immediate decrease in the level of high Mr CD45 mRNA after mitogen stimulation of T cells, quickly followed at 4 h by an increase to above steady state levels. This is consistent with the transitory increase in cell-surface density of CD45RA observed at 1 to 2 days poststimulation. Within 24 h of stimulation the level of high Mr CD45 mRNA declines precipitously such that little or no mRNA encoding any of the alternatively spliced exons is detectable. High levels of CD45 mRNA are detectable at later points but this does not hybridize with the Sfa N1 probe recognizing the abc exons suggesting that only the CD45RO mRNA splicing pattern is operative. We also show that CD45 mRNA have a relatively short t1/2. In mitogen-stimulated cells the t1/2 of high and low m.w. CD45 mRNA was estimated at 2.25 h and 3.5 h, respectively. In unstimulated T cells the t1/2 of high Mr CD45 mRNA was estimated at 2.8 h. CD45 mRNA is super-induced in the presence of the protein synthesis inhibitor, cycloheximide, indicating that the degradation and/or splicing of CD45 mRNA is controlled by a labile pathway, and suggesting that mechanisms may exist in vivo to prolong synthesis of CD45RA. The kinetics of accumulation for high Mr CD45 mRNA are very similar to those of the TCR beta-chain and pp56lck suggesting that these functionally linked signaling molecules are regulated in tandem. This implicates stringent molecular control mechanisms on the production of mRNA encoding either high or low m.w. isoforms, consistent with the fundamental role of CD45 in signal transduction, and the apparent need to selectively and sequentially express functionally distinct external CD45 domains.     

Uridine transport in basolateral plasma membrane vesicles from rat liver

Summary The characteristics of uridine transport were studied in basolateral plasma membrane vesicles isolated from rat liver. Uridine was not metabolized under transport measurement conditions and was taken up into an osmotically active space with no significant binding of uridine to the membrane vesicles. Uridine uptake was sodium dependent, showing no significant stimulation by other monovalent cations. Kinetic analysis of the sodium-dependent component showed a single system with Michaelis-Menten kinetics. Parameter values were K _M 8.9 m and V _max 0.57 pmol/mg prot/sec. Uridine transport proved to be electrogenic, since, firstly, the Hill plot of the kinetic data suggested a 1 uridine: 1 Na⁺ stoichiometry, secondly, valinomycin enhanced basal uridine uptake rates and, thirdly, the permeant nature of the Na⁺ counterions determined uridine transport rates (SCN^– > NO ₃ ^– > Cl^– > SO ₄ ^2– ). Other purines and pyrimidines cis-inhibited and trans-stimulated uridine uptake.This work has been partially supported by grant PM90-0162 from D.G.I.C.Y.T. (Ministerio de Educación y Ciencia, Spain). B.R.-M. is a research fellow supported by the Nestlé Nutrition Research Grant Programme.     

Effect of glutathione depletion on urinary acidification in the rat

Glutathione (GSH) depletion by diethyl maleate (DEM) administration and its rapid repletion were associated with the development of a moderate acidosis in the rat. The acidosis observed after DEM treatment could be a consequence of an impairment of lactate metabolism. GSH-depleted rats also showed an increased urine pH and a higher bicarbonate fractional excretion compared with control rats. Renal bicarbonate excretion was magnified when blood bicarbonate levels were normalized by means of a bicarbonate infusion in GSH-depleted rats; however, the amount of bicarbonate excreted in the urine was a very small fraction (less than 5%) of the calculated filtered load. GSH-depleted rats failed to elevate the relation urine minus blood (U-B) pCO2 as compared with control rats when they were subjected to a high bicarbonate load to the distal portions of the nephron. All these data were consistent with a distal renal tubular acidosis due to GSH depletion which could participate in the maintenance of the systemic acidosis, although it is unlikely that it is the primary cause of the acidosis.     

Power consumption of three impeller combinations in mixing Xanthan fermentation broths

Xanthan gum fermentation represents a good model for the study of the mixing of rheologically complex culture broths. Most of the previous work on power consumption dealt with ‘standard’, single impellers and used model fluids to simulate xanthan broths. This work describes the characterization of three dual-impeller combinations (D/T = 0·53) for the mixing of dehydrated—reconstituted fermentation broths of Xanthomonas campestris that had matched rheology to the actual broths. The bottom impeller was a Rushton turbine (RT) and the top impeller was another RT, a 45° pitched blade turbine (PT) or an A-310 Lightnin mixer (A310). The experiments were carried out in a tank of 0·0094 m³ working volume equipped with an air bearing dynamometer. The power was measured in a wide range of xanthan concentrations (5–40 kg m⁻³) in aerated (0·25, 0·5 and 1·0 vvm) and unaerated conditions. Unaerated power number (Po) vs. Reynolds number (Re) curves showed similar trends for the three combinations. Exponents close to −1 were obtained in the laminar region. A minimum in Po (Po_min) occurred at Re = 30–40, then increasing to a plateau value which was evident at Re> 200. In the transition region Po_min values were 4·3 (RT and RT), 3·6 (RT and PT) and 2·4 (RT and A310). The aerated power data for (RT and PT) and (RT and A-310) showed higher torque instabilities than the dual RT combinations at higher xanthan concentrations. The higher the xanthan concentrations, the higher the drop in power and the less important the effect of the aeration rate. Among the combinations tested, when using Rushton turbines, the well-mixed ‘cavern’ reached the tank wall (i.e., fluid motion was observed) at the lowest volumetric power input. High     

Effects of eel (Anguilla anguilla L.) removals from selected sites of a stream on its subsequent densities

We assessed the effect of eel (Anguilla anguilla) removal from three sites of a Cantabrian stream upon its subsequent densities. In the first sample (Sept. 1986) numbers and densities were estimated as 43, 45 and 84 ind and 3490, 3030 and 3750 ind ha ⁻¹. Removal of these eels reduced the subsequent numbers and densities which, except on two occasions, were never reached again during the two years (eleven estimates) of study. Highest densities were recorded in the uppermost site in May and July, 1987, coincident with a strong drought and the lowest densities occurred in 1988 during a normal wet year. We hypothesize first that, because of a selective underground homing behaviour of eels, electro-fishing is inefficient and results in underestimates of the population. Second, seasonal variations of water discharge and droughts may not influence the homing behaviour of'eels until a threshold of dryness is reached. If this occurs, eels abandon their refuges and move towards the stream bottom. It seems that in Arroyo Chabatchos this threshold was exceeded in the summer of 1987 when the highest densities were estimated. The re-colonization of these sites experimentally depleted of eels, is a slow procces that lasts for, at least, two years.