Kinetics of the activation of rat liver pyruvate kinase by fructose 1,6-disphosphate and methods for characterizing hysteretic transitions

1. Kinetics of fructose 1,6-diphosphate activation of liver pyruvate kinase type I inhibited with MgATP and l-alanine are described as a function of enzyme and fructose 1,6-diphosphate concentrations. These results can be explained by a single pseudo-first-order transition of the enzyme into an active form, independent of the enzyme concentration. This rate constant, k(app.)=0.24s(-1) with 0.02mm-fructose 1,6-diphosphate (t(0.9) approximately 10s where t(0.9) is the time for 90% conversion), is an increasing function of fructose 1,6-diphosphate concentration far beyond that needed to maximally activate enzyme equilibrated with fructose 1,6-diphosphate (about 20mum). 2. The model equations are best analysed with numerical techniques which are described. These techniques are useful in studying similar slow transients frequently observed in stopped-flow studies of enzymes. 3. Shorter transients (t(0.9)=0.5-1.5s) were observed in the kinetic response of the enzyme to the addition of MgATP or phosphoenolpyruvate, but were not further characterized.     

Interspecific incompatibility due to stylar barriers in tuber-bearing and closely related non-tuber-bearing Solanums

Summary The phenomenon of interspecific incompatibility between various wild tuber-bearing and closely related non-tuber-bearing Solanum species was studied. One area of investigation included an examination of possible protein interactions in the incompatibility reaction using SDS electrophoresis. Pollen tube inhibition and morphology were examined in conjunction with biochemical analysis. Two sets of crosses were examined: interspecific tuber-bearing species crosses and interspecific tuber-bearing × non-tuber-bearing species crosses. These crosses had consistent pollen tube inhibition in the upper one-third of the style. The upper third of the styles of incompatibly pollinated, compatibly pollinated, and unpollinated styles was studied under fluorescence microscopy to observe pollen tube growth and morphology. Interspecific tuber-bearing × non-tuber-bearing species crosses demonstrated consistent pollen tube inhibition just below the stigma with frequent pollen tube swelling and bursting and extensive callose deposition along the pollen tube wall. Interspecific tuber-bearing species crosses had pollen tube inhibition further down the style with pollen tube tip tapering and extensive callose deposition. Stylar proteins of the lower two-thirds of the styles were analyzed with SDS electrophoresis. No unique protein differences were found to be specifically associated with the interspecific incompatibility reaction in this portion of the style.Cooperative investigation of the U.S. Department of Agriculture, Agricultural Research Service, and the Wisconsin Experiment Station. Supported in part by the USDA, Cooperative States Research Service Competitive grant no. 83-CRCR-1-1253     

Calcium- Versus G Protein-Mediated Phosphoinositide Hydrolysis in Rat Cerebral Cortical Synaptoneurosomes

The role of calcium and sodium in stimulating phosphoinositide hydrolysis in brain was investigated in rat cerebral cortical synaptoneurosomes. In buffer containing 136 mM sodium and various concentrations of added calcium (0-1.0 mM), basal, potassium-stimulated, and norepinephrine-stimulated formation of 3H-inositol phosphates decreased with decreasing extracellular calcium. Potassium- and norepinephrine-stimulated formation of 3H-inositol phosphates was reduced to basal levels by addition of EGTA. Isosmotically replacing sodium with choline chloride or N-methyl-D-glucamine to disrupt Na+/Ca2+ exchange resulted in a large increase in the formation of 3H-inositol phosphates. Measurement of cytosolic calcium with fura-2 revealed that the cytosolic calcium concentration was sensitive to changes in the extracellular calcium concentration and increased on resuspension of synaptoneurosomes in sodium-free rather than sodium-containing medium. In the absence of sodium, potassium-stimulated formation of 3H-inositol phosphates was reduced or eliminated, depending on the extracellular calcium concentration. Subtraction of basal formation of 3H-inositol phosphates from that in the presence of 1 mM carbachol or 100 microM norepinephrine revealed that the carbachol-stimulated component was the same in the presence and absence of sodium, whereas the norepinephrine-stimulated component was reduced in the absence of sodium. Addition of the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate inhibited norepinephrine- and, to a lesser extent, carbachol but not basal or aluminum fluoride-stimulated formation of 3H-inositol phosphates in sodium-free medium. These results suggest that an increase in intracellular calcium, via disruption of Na+/Ca2+ exchange or depolarization-induced calcium influx, may explain previous demonstrations that agents that stimulate Na+ influx can also stimulate phosphoinositide hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fetal cells in maternal blood: recovery by charge flow separation

Fetal blood cells can be recovered from the maternal circulation by charge flow separation (CFS), a method that obviates the risks associated with amniocentesis and chorionic villus sampling. By CFS, we processed blood samples from 13 women carrying male fetuses, 2 carrying fetuses with trisomy 21, and 1 who had delivered a stillborn infant with trisomy 18. On average more than 2000 fetal nucleated red blood cells were recovered per 20-ml sample of maternal blood. Recovery of fetal cells was confirmed by fluorescence in situ hybridization with probes for chromosomes Y, 18 and 21. After culturing of CFS-processed cells, amplification by the polymerase chain reaction revealed Y-chromosomal DNA in clones from four of six women bearing male fetuses, but not in clones from three women bearing female fetuses. Received: 8 January 1996 / Revised: 22 March 1996     

Developmental and genotypic differences in the response of pea stem segments to auxin

The objective of this investigation was to examine the response to exogenous auxin (indole-3-acetic acid; IAA)of stem segments at two developmental stages. The standard auxin response of excised stem segments and intact plants consists of an initial growth response and a prolonged growth response. We found that this biphasic response does not occur in internodes at very early stages. Stem segments of light grown pea of various genotypes were cut when the fourth internode was at 6–13% of full expansion (early-expansion) or at 18–25% of full expansion (mid-expansion). Length measurements of excised segments were made after 48 hours of incubation on buffer with or without auxin. An angular position transducer linked to a computerized data collection system provided high-resolution measurement of growth of stacks of segments incubated in buffer over 20 hours. Early-expansion segments of all genotypes deviated from the standard auxin response, while mid-expansion segments responded in a manner consistent with previous reports. Early-expansion segments of tall, light-grown plants were unique in showing an auxin-induced inhibition of growth. The auxin-induced inhibition correlated with high endogenous auxin content, as determined by HPLC and GC/MS, across genotypes and between early-expansion and mid-expansion segments of tall plants. Measurement of ethylene evolved from stem segments in response to auxin, and treatment of segments with the ethylene action inhibitor, norbornadiene, showed the inhibition to be mediated in part by heightened ethylene sensitivity. Growth of early-expansion segments of dwarf and severe dwarf plants was stimulated by exogenous auxin, but the growth rate increase was delayed compared to that in mid-expansion segments. This is the first time that such a growth response, termed the delayed growth response has been emonstrated. It is concluded that developmental stage and endogenous hormone content affect tissue response to exogenous auxin.     

Effects of exercise on mammary metabolism in the lactating ewe

Mammary metabolism in multiparous lactating ewes fed either lucerne chaff:barley grain (L:B; 70:30) or lucerne chaff:lupin grain (L:Lu; 70:30) diets was measured while at rest, during exercise on a treadmill at 0.7 m s⁻¹ on a 10 ° slope for 60 min, and during 30 min recovery from exercise. The effects of these treatments on plasma glucose, lactate, alpha-amino nitrogen (-amino N), non-esterified fatty acids (NEFA) and acetate were measured. Net mammary uptake of oxygen and metabolites was calculated from mammary blood flow and arteriovenous concentration (A − V) differences.

Mammary blood flow was reduced by 25% during exercise. Arterial concentrations of oxygen, glucose, lactate, -amino N and NEFA increased during exercise, whereas acetate concentration either remained unchanged or declined. Mammary A − V differences were significantly higher for oxygen, glucose, lactate and NEFA, and tended to be higher for -amino N and lower for acetate during exercise. The mammary uptakes of oxygen, glucose, lactate and -amino N were unaffected by exercise, whereas the uptake of NEFA was significantly increased and that of acetate was significantly reduced. The changes in arterial concentrations and mammary uptakes in response to exercise were not significantly affected by the diet. The responses in acetate and NEFA fluxes across the mammary gland might bring a change in the utilization of other metabolites as well as in the fatty acid composition of milk fat.     

Detachment of transformed cells

A parallel-plate flow chamber was used to quantify the detachment of normal cloned rat embryo fibroblasts (CREF) fibroblasts,ras-transformed CREF fibroblasts (CREF T24), and CREF T24 fibroblasts transfected with a Krev/RAP1A suppressor gene (HK B1) from a confluent monolayer of normal CREF fibroblasts to determine if the expression patterns of CD44 variants (mol wt 110 and 140 kDa) corresponded with detachment properties and metastatic potential. In the detachment assay, known shear stresses ranging from 20–24 dyn/cm² were applied to the adherent cells and the number of cells detached from the monolayer after 180 s was determined. Results showed that cellular expression of CD44 variants correlated with the metastatic potential of the cells and with the cells’ ability to detach from a monolayer of normal cells. Western blot analysis showed a low level of expression of the CD44 variants in the normal cell line, CREF, and the lowly metastatic cell line, HK B1. Detachment studies showed a low percentage of detachment of both of these cell lines from a normal cell monolayer. Tumor-derived (HK B1-T) and lung nodule-derived (HK B1-M) cell lines were established and both formed tumors and metastasis with reduced latency periods as compared to HK B1, but still showed a markedly delayed latency period compared to the highly metastatic cell line, CREF T24. Both of these cell lines showed a higher expression of the CD44 variants as compared to CREF and HK B1, and detached easier than CREF and HK B1. CREF T24 showed a much higher level of expression of the variants and had a higher percentage detachment than all other cell lines. To further test the role of the CD44 variants in the ability of the cells to detach from the normal monolayer, CREF cells were transfected with a DNA construct that constitutively expresses the CD44 variants and the detachment properties of three randomly selected clones were studied. Clones 2 and 3 showed a low level of expression of the CD44 variants after transfection and detached from the normal monolayer similar to CREF. Clone 1 showed a high level of expression of the CD44 variants and the detachment of these cells was significantly higher than CREF. From these results, it is concluded that in the five cell lines studied, expression of the CD44 variants play a significant role in the ability of the cells to detach from a monolayer of normal cells. It is hypothesized that this detachment may be an important component of a cell’s ability to metastasize.