Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.     

Effect of interferons on protein synthesis in human lymphocytes: enhanced synthesis of eight specific peptides in T cells and activation-dependent inhibition of overall protein synthesis

Lymphoblastoid and fibroblast IFN inhibited PHA stimulation of overall protein synthesis in human lymphocytes by ca. 30%. Inhibition occurred within the first 6 hr of PHA treatment and was not progressive. DNA synthesis at 48 hr was inhibited to the same extent. Overall protein synthesis in resting lymphocytes was not detectably inhibited by IFN concentrations up to 1000 U/ml. Thus, inhibition of protein synthesis and subsequent reduction of cell proliferation by IFN require certain early events in mitogen activation. Resting lymphocytes were not unresponsive to IFN treatment, however. Two-dimensional electrophoretic analysis of newly synthesized proteins after IFN treatment showed enhanced synthesis of a specific set of eight peptides (I-peptides), which were shown to be synthesized in T lymphocytes. This enhancement was produced by both IFN-alpha and IFN-beta after 4 to 6 hr of exposure and was identical for all lymphocyte donors studied. After growth stimulation, IFN treatment produced no enhancements of additional peptides, although the original eight I-peptides were enhanced as usual. It is concluded that the biochemical activities of the I-peptides, which remain to be determined, cannot inhibit protein synthesis in resting lymphocytes, but may do so after mitogen activation, when the major physiologic restriction of lymphocyte protein synthesis is released. Alternatively, the I-peptides may be unrelated to regulation of protein synthesis but may be involved in viral protection or enhancement of NK activity.     

Major gene analysis of quantitative variation in blood clotting factor X levels.

Blood clotting factor ten (X) levels measured in 149 people in six pedigrees were found to fit a mixture of normal distributions. No environmental effect could be identified to account for the wide separation in the means of these distributions. Pedigree analysis reveals that the data are compatible with an autosomal, one locus, two allele genetic model affecting factor X activity. Goodness of fit tests suggest that the allele for low levels of factor X is dominant, though on the basis of likelihood tests, mean heterozygote levels are different from mean homozygote levels. A similar bimodal distribution for factor X levels observed previously in a separate sample of 207 young men, indicated that the proposed dominant allele has an estimated population gene frequency of .53. The earlier estimate is remarkably similar to that obtained with the currently ascertained pedigrees. The postulated major gene accounts for more than half of the variation in factor X levels.     

Isolated Aortocoronary Bypass Operations in Patients Over 70 Years of Age: A Comparison With Results in Patients 50 to 59 Years Old

The early and late morbidity, mortality and beneficial effects of isolated aortocoronary bypass operations in a group of 35 patients 70 years old or older were compared with those factors in patients 50 to 59 years old. The patients in both groups were matched according to the year in which the operation was done and the number of vessels bypassed. Left ventricular function, estimated by the angiographically calculated ejection fraction, was not statistically different in the two groups. Cardiac index, while adequate in both groups, was significantly lower in the older age group. Comparisons were made of “early” events, such as perioperative myocardial infarction, perioperative death and length of post-operative hospital stay; and of “late” events, including myocardial infarction, angina pectoris, congestive heart failure and death, which occurred after patients were discharged from the hospital. The mean length of follow-up of patients was similar in both groups.In comparing early events in the two groups, there was no statistically significant difference in the incidence of perioperative myocardial infarction, perioperative mortality or mean length of postoperative hospital stays. With regard to late events, there was no statistically significant difference in the incidences of myocardial infarction, angina pectoris or mortality.     

Mass balance of heavy metal uptake by encapsulated cultures ofKlebsiella aerogenes

Dialysis was employed as a method of speciating heavy metals in cultures of an extracellular polymer forming strain ofKlebsiella aerogenes. A noncapsulated strain of the same bacterium was used as a control, and a mass balance of copper, cadmium, cobalt, nickel, and manganese in batch culture at pH 4.5 and pH 6.8 and in continuous culture at pH 6.8 was constructed. Copper and cadmium were accumulated by the cell during rapid proliferation whereas all 5 metals were bound nonspecifically by extracellular polymer produced during stationary phase and at low dilution rates. The presence of extracellular polymer appeared to inhibit cellular uptake of nickel. At the lower pH, metal uptake was considerably reduced. The results are discussed in the context of metal removal in the activated sludge process of waste water treatment.     

Synthesis and characterization of 5-azido-UDP-glucuronic acid. A new photoaffinity probe for UDP-glucuronic acid-utilizing proteins.

A new active site-directed photoaffinity analogue, [beta-32P]5-azido-UDP-glucuronic acid (UDP-GlcA), was enzymatically synthesized from [beta-32P]5-N3UDP-Glc using UDP-glucose dehydrogenase. The product was characterized by its mobility on ion exchange and two thin-layer chromatographic systems, by its UV absorbance at 288 nm, and the loss of this absorbance after UV irradiation of the compound. Photoincorporation of [beta-32P]5-N3UDP-GlcA into bovine liver UDP-Glc dehydrogenase (EC 1.1.1.22) was saturable with an apparent Kd of 12.5 microM, and was inhibited by the known active-site effectors UDP-GlcA, UDP-Glc, and UDP-xylose. When human liver microsomes with known UDP-glucuronosyltransferase (EC 2.4.1.17) activities were photolabeled with [beta-32P]5-N3UDP-GlcA, major photolabeled bands of 35-37 and 50-54 kDa were detected. When rat liver microsomes from phenobarbital-injected rats were photolabeled with [beta-32P]5-N3UDP-GlcA, there was a marked increase in photoincorporation of a 51-kDa protein as compared with control animals. Evidence is presented which suggests that the photolabeled 51-54-kDa proteins in the liver microsomes from both tissues are UDP-glucuronosyltransferase and that [beta-32P]5-N3UDP-GlcA represents a new alternative approach in the study of UDP-glucuronosyltransferase and other UDP-GlcA-utilizing enzymes.     

Lipid vesicles which can bind to protein kinase C and activate the enzyme in the presence of EGTA.

Maximal protein kinase C activity with vesicles of phosphatidic acid and 1,2-dioleoyl-sn-glycerol is observed in the absence of added Ca2+. Addition of phosphatidylcholine to these vesicles restores some calcium dependence of enzyme activity. 1,2-Dioleoyl-sn-glycerol eliminates the Ca(2+)-dependence of protein kinase C activity found with phosphatidic acid alone. Phorbol esters do not mimic the action of 1,2-dioleoyl-sn-glycerol in this respect. This suggests that the 1,2-dioleoyl-sn-glycerol effect is a result of changes it causes in the physical properties of the membrane rather than to specific binding to the enzyme. The effect of 1,2-dioleoyl-sn-glycerol on the phosphatidic-acid-stimulated protein kinase C activity is dependent on the molar fraction of 1,2-dioleoyl-sn-glycerol used and results in a gradual shift from Ca2+ stimulation at low 1,2-dioleoyl-sn-glycerol concentrations to calcium inhibition at higher concentrations of 1,2-dioleoyl-sn-glycerol. Phosphatidylserine-stimulated activity is also shown to be largely independent of the calcium concentration at higher molar fractions of 1,2-dioleoyl-sn-glycerol. Thus, with certain lipid compositions, protein kinase C activity becomes independent of the calcium concentration or requires only very low, stoichiometric binding of Ca2+ to high affinity sites on the enzyme. Protein kinase C can bind to phosphatidic acid vesicles more readily than it can bind to phosphatidylserine vesicles in the absence of calcium. Addition of 1,2-dioleoyl-sn-glycerol to phosphatidylserine vesicles promotes the partitioning of protein kinase C into the membrane in the absence of added Ca2+. There is no isozyme specificity in this binding. These results suggest that a less-tightly packed headgroup region of the bilayer causes increased insertion of protein kinase C into the membrane. This is a necessary but not sufficient condition for activation of the enzyme in the presence of EGTA.     

Application of 5-azido-UDP-glucose and 5-azido-UDP-glucuronic acid photoaffinity probes for the determination of the active site orientation of microsomal UDP-glucosyltransferases and UDP-glucuronosyltransferases.

A new approach to determining the active site orientation of microsomal glycosyltransferases is presented which utilizes the photoaffinity analogs [32P]5-Azido-UDP-glucose ([32P]5N3UDP-Glc) and [32P]5-Azido-UDP-glucuronic acid ([32P]5N3UDP-GlcA). It was previously shown that both photoprobes could be used to photolabel UDP-glucose:dolichol phosphate glucosyltransferase (Glc-P-Dol synthase), as well as the family of UDP-glucuronosyltransferases in rat liver microsomes. The effects of detergents, proteases, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on the photolabeling of these enzymes were examined in intact rat liver microsomes. Photolabeling of Glc-P-Dol synthase by either photoprobe was the same in intact or disrupted vesicles, was susceptible to trypsin digestion, and was inhibited by the nonpenetrating inhibitor DIDS. Photolabeling of the UDP-glucuronosyltransferases by [32P]5N3UDP-GlcA was stimulated 1.3-fold in disrupted vesicles as compared to intact vesicles, whereas photolabeling of these enzymes by [32P]5N3UDP-Glc showed a 14-fold increase when vesicles were disrupted. Photolabeled UDP-glucuronosyltransferases were only susceptible to trypsin digestion in disrupted vesicles, and this was further verified by Western blot analyses. The results indicate a cytoplasmic orientation for access of UDP-sugars to Glc-P-Dol synthase and a lumenal orientation of most UDP-glucuronosyltransferases.     

Discrimination and consistency of five myosin ATPase stains in human normal and duchenne dystrophic muscle

Summary Limitations in the ability of the human visual system to assess accurately the relative staining densities of individual fibers in muscle tissue stained for myosin ATPase can complicate the objective evaluation of fiber type populations. In this study a novel approach is employed which utilizes human visual capabilities to provide accurate fiber classification. Using this approach, the ability of five ATPase staining techniques to discriminate fiber type categories in single samples of human normal and Duchenne dystrophic skeletal muscle is evaluated, as is the consistency of the fiber type classifications between stains. While no major discrepancies in fiber typing were observed in the sample of normal muscle, significant differences in classification, along with a decrease in the ability to discriminate fiber types were noted in the sample of Duchenne muscular dystrophy. For the most part, these discrepancies were resolved by a re-interpretation of the staining characteristics of fibers in one stain.This work was supported in part by NIH grant NS 15584, and by a grant from the Muscular Dystrophy Association