Mechanisms of cytoskeletal regulation: functional and antigenic diversity in human erythrocyte and brain beta spectrin

A study of human erythrocyte and brain spectrin with particular emphasis on the beta subunits revealed a structural homology but functional dissimilarity between these two molecules. Six monoclonal antibodies raised to human erythrocyte beta spectrin identify three of the four proteolytically defined domains of erythrocyte beta spectrin. Five of these monoclonal antibodies cross-react with human brain spectrin. None of a previously identified set of alpha erythrocyte spectrin monoclonal antibodies [Yurchenco et al: J Biol Chem 257:9102, 1982] reacted with brain spectrin. A domain map generated by limited tryptic digestion shows that brain spectrin is composed of proteolytically resistant domains analogous to erythrocyte spectrin, but the brain protein is more basic. The binding of brain spectrin to erythrocyte ankyrin, both in solution and on erythrocyte IOVs, yielded an association constant approximately 100 time weaker than for erythrocyte spectrin. The binding of azido-calmodulin under native conditions was specific for the erythrocyte beta subunit but was not calcium dependent. In contrast, azido-calmodulin bound only to the alpha subunit of brain spectrin in a calcium-dependent manner. The similarity of structure but modified functional characteristics of the brain and erythrocyte beta spectrins suggest that these proteins serve different cellular roles.     

Relationship between intermediate filaments and microfilaments in cultured fibroblasts: evidence for common foci during cell spreading

Spreading and fully spread chick embryo fibroblasts (CEF) were examined by double-label fluorescence microscopy using the actin-specific probe rhodamine-phalloidin and an antibody directed against CEF intermediate filaments (IF). During midspreading, a striking relationship became discernible: statistical analysis showed that approximately half of the cell population exhibited one or more phase-dense, phalloidin-binding nodules that appeared to act as foci from which IF diverged. Coincidence between actin-containing structures and IF was not limited to these centers; IF could also frequently be seen running in close parallel arrays with stress fibers. Ultrastructural analysis confirmed the presence of non-membrane-bound out-pocketings along the length of stress fibers from which 10-nm IF diverged. These structures varied in size and shape, and displayed a dense, fine fibrillar appearance. IF and microfilaments (MF) were distinguished by size and by decoration of MF with myosin subfragment-1. Other IF-MF interactions were seen in cells of all stages: IF were observed to loop through stress fibers, most frequently at the cell margins. In colchicine-treated cells, IF became redistributed into cables that often ran parallel and appeared to merge with stress fibers. Cytochalasin D-treated CEF exhibited loose aggregates of actin-containing material that appeared to be associated with IF. These results suggest the possibility of an interaction between actin-containing structures and IF, particularly during cell spreading in cultured fibroblasts.     

Role and location of NAD malic enzyme in thermogenic tissues of Araceae

This work was done to discover how those nonphotosynthetic tissues of the Araceae that become thermogenic release, as CO2, carbon recently fixed by phosphoenolpyruvate carboxylase. Extracts of clubs of the spadix of Arum maculatum showed no activity for phosphoenolpyruvate carboxykinase and low activities of NADP malic enzyme. NAD malic enzyme activity in the above extracts and in those of thermogenic tissues of other Araceae was appreciable. Analysis of homogenates of clubs of Typhonium giraldii by differential centrifugation and sucrose gradients showed that NAD malic enzyme was confined to mitochondria. Centrifugation of mitochondria after freezing and thawing left all the NAD malic enzyme in the supernatant. NAD malic enzyme in isolated, intact mitochondria was completely latent, and was completely protected from exogenous trypsin. The responses of this latency and protection to different concentrations of Triton X-100 suggested that none of the NAD malic enzyme was accessible from either the outside or the intermembrane space of the mitochondria. Treatment of excised clubs of A. maculatum with 2-N-butylmalonate largely prevented the development of the rapid respiration responsible for thermogenesis, and severely inhibited dark fixation of 14CO2. The conclusion is that in mature clubs of the Araceae phosphoenolpyruvate is converted to malate in the cytosol by phosphoenolpyruvate carboxylase and NAD malate dehydrogenase, and that this malate then enters the mitochondrial matrix where it is converted to pyruvate by NAD malic enzyme.     

Fast atom bombardment: evidence of disproportionation and recombination of a synthetic porphyrin in the matrix

In addition to dimerization and polymerization of samples as previously suggested, it appears that during FAB-MS, reactions in the sample matrix can occur to yield new compounds that are recombinations of molecular fragments. This type of reaction may be especially critical to the integrity of peptide sequencing using FAB, since the reactions cited in this report involve the formation and rupture of amides or peptide bonds.     

Effects of oral propranolol and exercise protocol on indices of aerobic function in normal man

The exercise responses to two different progressive, upright cycle ergometer tests were studied in nine healthy, young subjects either with no drug (ND) or following 48 h or oral propranolol (P) (40 mg q.i.d.). The ergometer tests increased work rate by 30 W either every 30 s or every 4 min. Propranolol caused a significant (p less than 0.05) reduction in peak oxygen uptake (VO2) during both the 30-s and 4-min tests (30-s ND, 3949 +/- 718 mL X min-1 (means +/- SD); 30-s P, 3408 +/- 778 mL X min-1; 4-min ND, 4058 +/- 409 mL X min-1; 4-min P, 3725 +/- 573 mL X min-1). There was no difference between 30-s ND and 4-min ND for peak VO2. The ventilatory anaerobic threshold was not significantly different between any test (30-s ND, 2337 +/- 434 mL O2 X min-1; 30-s P, 2174 +/- 406 mL O2 X min-1; ND, 2433 +/- 685 mL O2 X min-1; 4-min P, 2296 +/- 604 mL O2 X min-1). The VO2 at which blood lactate had increased by 0.5 mM above resting levels was significantly lower than the ventilatory anaerobic threshold for the 4-min ND (1917 +/- 489) and the 4-min P (1978 +/- 412) tests, but was not different for the 30-s ND and 30-s P tests. At exhaustion in the progressive tests, the blood PCO2 was higher (p less than 0.05) in both 30-s tests than 4-min tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial NH2- and COOH-terminal sequence and cyanogen bromide peptide analysis of Escherichia coli sn-glycerol-3-phosphate acyltransferase

The sn-glycerol-3-phosphate acyltransferase from Escherichia coli, an integral membrane protein whose activity is dependent on phospholipids, was purified to near homogeneity (Green, P. R., Merrill, A. H., Jr., and Bell, R. M., (1981) J. Biol. Chem. 256, 11151-11159). Determination of a partial NH2-terminal sequence and the COOH terminus permitted alignment of the polypeptide on the sequenced sn-glycerol-3-phosphate acyltransferase structural gene (Lightner, V. A., Bell, R. M., and Modrich, P. (1983) J. Biol. Chem. 258, 10856-10861). Processing of the sn-glycerol-3-phosphate acyltransferase is apparently limited to the removal of the NH2-terminal formylmethionine. Thirteen of 27 possible cyanogen bromide peptides predicted from the DNA sequence were purified, characterized, and assigned to their location in the primary structure. Three peptides located at positions throughout the sequence were partially sequenced by automated Edman degradation. The partial sequence analysis of the homogeneous sn-glycerol-3-phosphate acyltransferase is fully in accord with the primary structure inferred from the DNA sequence.     

A comparison of the agar cloning and microtitration techniques for assaying cell survival and mutation frequency in L5178Y mouse lymphoma cells

Microtitration methods for assaying cell survival and mutation frequency to ouabain resistance, 6-thioguanine resistance and 1-β-d-arabinofuranosyl cytosine resistance in L5178Y mouse lymphoma cells were compared to the standard agar cloning technique. The two methods gave essentially similar results for untreated cells, and after treatment with ethyl methanesulphonate and 4-nitroquinoline 1-oxide. Potential advantages of the microtitration method as a routine assay system are discussed.     

Properties of growth hormone receptors in relation to the adipose conversion of 3T3 cells

Cultured preadipose 3T3 cells undergo a process of differentiation in which they convert to adipose cells. Growth hormone promotes this conversion. Since 3T3 sublines vary in their susceptibility to adipose conversion, it was of interest to examine the properties of the growth hormone receptors in relation to that susceptibility. It was found that preadipose 3T3-F442A cells, which are able to convert to adipose cells with high frequency, are able to bind about 10(4) growth hormone molecules per cell with Kd approximately 10(-9) M. After adipose conversion, no appreciable change in hormone binding was detected. The binding of growth hormone to 3T3-C2 cells (a line virtually insusceptible to adipose conversion) was indistinguishable from that to 3T3-F442A cells. Internalization and degradation of the hormone were also similar in the two cell lines. Susceptibility to adipose conversion is therefore not determined by the relative ability of the cells to bind or degrade the hormone, but must instead depend on some response, as yet unidentified, that follows binding of the hormone.     

A Tipula paludosa population with a high incidence of two pathogens

The incidence of lethal parasites in the larvae of a Tipula paludosa population was monitored for two seasons. The proportions of larvae infected with Tipula iridescent virus (TIV) and a tachinid insect were similar to those in previously studied populations, whereas the proportions of larvae infected with Tipula nuclear polyhedrosis virus (NPV) and a spore-forming bacterium (SFB) were higher. Conservative estimates of mortality due to these four agents were 10.7% in 1977–1978 and 7.7% in 1978–1979. The mean population density and the proportion of SFB-infected larvae were lower in 1978–1979 than in 1977–1978, while the proportion of NPV-infected larvae was higher. In 1979 the proportion of NPV-infected larvae was positively correlated with population density, which was highest in the wettest part of the study area. In both seasons the proportion of SFB-infected larvae was negatively correlated with population density. Larvae infected with the NPV or the SFB became pallid at an advanced stage of infection, but, although infected larvae were found throughout the larval period, pallid larvae were only found in the later part. It is suggested that larvae become infected in an early instar, then the infections slowly develop throughout the remainder of the larval period. Five larvae were found with mixed infections; four were infected with the SFB and NPV, while the fifth was infected with the SFB and TIV.