Biomedical subjects
Publications and source records attributed to M Bloom.
This first observation of the deuterium nuclear magnetic resonance (2H-NMR) spectrum of phospholipid molecules incorporated into intact human erythrocyte ghosts shows that the liquid crystalline phase is stable down to a temperature of -5 degrees C. The quality of the 3H-NMR spectra indicate that it is now possible to carry out clinical studies of erythrocyte membranes using the techniques employed in this study.
Over a three-year period, we have seen in consultation four children whose mothers cited complaints referable to every organ system and which had persisted for many years. The parents had consulted a total of 99 physicians in eight states. Absence from school ranged from 40 to 200 days a year. Physical examinations of all patients and extensive and repeated laboratory studies were normal. On psychiatric examination the mothers exhibited paranoid thinking and a conviction of serious medical illness in their child which approached delusional proportions. They resisted psychiatric consultation and refused psychotherapy. The mother-child relationship was remarkably symbiotic, the two teen-age patients essentially voicing complaints which were indistinguishable from those reported by their mothers. The fathers invariably supported their wives' concerns. Subsequently, parents and children left treatment, continuing to "doctor shop." Long-standing multisystem complaints in a child with normal growth and maturation are incompatible with any known significant organic disease, but suggest a serious emotional problem within the family. Further, parents who take such children from doctor to doctor are frequently disturbed themselves and may use an offspring as a proxy patient. An accurate diagnosis depends on careful history-taking from parents, patient, health professionals, and schools.
Ribulose bisphosphate carboxylase consists of cytoplasmically synthesized "small" subunits and chloroplast-synthesized "large" subunits. Large subunits of ribulose bisphosphate carboxylase synthesized in vivo or in organello can be recovered from intact chloroplasts in the form of two different complexes with sedimentation coefficients of 7S and 29S. About one-third to one-half of the large subunits synthesized in isolated chloroplasts are found in the 7S complex, the remainder being found in the 29S complex. Upon prolonged illumination of the chloroplasts, newly synthesized large subunits accumulate in the 18S ribulose bisphosphate carboxylase molecule and disappear from both the 7S and the 29S large subunit complexes. The 29S complex undergoes an in vitro dissociation reaction and is not as stable as ribulose bisphosphate carboxylase. The data indicate that (a) the 7S large subunit complex is a chloroplast product, the (b) the 29S large subunit complex is labeled in vivo, that (c) each of these two complexes can account quantitatively for all the large subunits assembled into RuBPCase in organello, and that (d) excess large subunits are degraded in chloroplasts.
It has generally been assumed that CLL B cells do not contain cytoplasmic immunoglobulin (Cylg), although these cells express surface membrane immunoglobulin (Smlg). The present study, in which Smlg and Cylg determinations were performed simultaneously using leukemia B cells from 20 patients with CLL, demonstrates that Cylg staining was detectable in each CLL cell population examined and that the intensity of cytoplasmic immunofluorescent staining in each instance was moderate to strong. Both Smlg and Cylg light chain determinations indicated monoclonality in all 20 cases. The heavy chain class of Smlg and Cylg in each CLL cell population was not uniformly comparable, however. The majority (15 cases) of CLL B cells contained mu heavy chain Cylg and mu and delta heavy chain Cylg was demonstrated in the remaining 6 cases. In contrast, the Smlg phenotypes appeared heterogeneous with both gamma and alpha chain determinants found associated with mu or mu and delta chains on the same leukemia cell populations. This apparent polyclonal Smlg staining pattern was most likely due to nonspecific adsorption of the patient's own serum Ig by Fc receptors on CLL B cells. It is concluded that the great majority of CLL B cells contain detectable Cylg and that Cylg determination is superior to Smlg phenotyping in documenting the monoclonality of CLL.
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2H NMR and EPR spectra have been obtained as a function of temperature and protein concentration from the same samples of beef heart cytochrome c oxidase reconstituted into 1-(16,16,16-trideuteriopalmitoyl)-2-palmitoleoyl-sn-glycero-3-phosphocholine. At all temperatures, the EPR spectra show the characteristic "bound" and "free" components, while the 2H NMR spectra show only a narrow distribution of orientational order parameters. At temperatures near the phase transition of the pure lipid, the dependence of the 2H NMR average orientational order on protein concentration fits a two-stage model in which the phospholipid molecular exchange rapidly between two states tentatively identified as sites either on or off the protein surface. From this model, the 2H NMR spectra yield a value of 0.18 mg of phospholipid per mg of protein as necessary to cover the surface of cytochrome c oxidase, which is the same value as derived from the EPR spectra at -20 degrees C. Both the 2H NMR and EPR spectra vary markedly with temperature. At temperatures well above the phase transition of the pure lipid, the average orientational parameters derived from the 2H NMR spectra are independent of protein concentration and are the same as for the lipid alone. Qualitatively, the EPR spectra show large apparent decreases in the average orientational order with increasing temperature. Analysis of 2H NMR relaxation rates indicates an additional motion in the presence of protein with a correlation time of 10(-6)-10(-7) s. If this new motion is associated with exchange between the two states, a minimum value of 10(6)-10(7) s-1 for the exchange rate is obtained, assuming that the lipids on the protein surface are much more motionally restricted than the rest of the lipid. Such an exchange rate is compatible with the observed differences in 2H NMR and EPR spectra. These results are consistent with short-lived, energetically weak interactions between cytochrome c oxidase and the phospholipids used in this study.
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2H nuclear magnetic resonance (NMR) of Acholesplasma laidlawii membranes grown on a medium supplemented with perdeuterated palmitic acid shows that at 42 degrees C or above, the membrane lipids are entirely in a fluid state, exhibiting the characteristic 'plateau' in the variation of deuterium quadrupolar splitting with chain position. Between 42 and 34 degrees C there is a well-defined gel-to-fluid phase transition encompassing the growth temperature of 37 degrees C, and at lower temperatures the membranes are in a highly ordered gel state. The 2H-NMR spectra of the gel phase membranes are similar to those of multilamellar dispersions of chain perdeuterated dipalmitoyl phosphatidylcholine (Davis, J.H. (1979) Biophys. J. 27, 339) as are the temperature dependences of the spectra and their moments. The incorporation of large amounts of cholesterol into the membrane removes the gel to fluid phase transition. Between 20 and 42 degrees C, the position dependence of the orientational order of the hydrocarbon chains of the membranes is similar to that of the fluid phase of the membranes without cholesterol, i.e., they exhibit the plateau in the deuterium quadrupolar splittings. However, the cholesterol-containing membranes have a higher average order, with the increases in order being greater for positions near the carbonyl group of the acyl chains. Below 20 degrees C the 2H spectra of the membranes containing cholesterol change dramatically in a fashion suggestive of complex motional and/or phase behaviour.
Specifically deuterated palmitic acid was incorporated into the membrane phospholipids of the L51 strain of Escherichia coli. The cytoplasmic and outer membranes were separated by using standard techniques and studied by deuterium nuclear magnetic resonance between 0 and 40 degrees C. Distinctive liquid-crystalline and gel spectra were observed to coexist over a wide temperature range. The relative intensities of these spectra provided a direct measure of the fraction of the deuterium-labeled phospholipids in the fluid state as a function of temperature. Above 37 degrees C, the amount of immobilized or gel-phase phospholipid is estimated to be less than 3% of the total phospholipid. The gel to liquid-crystalline transition region for the outer membrane was shifted upwards by approximately 7 degrees C relative to that of the cytoplasmic membrane, in agreement with previous studies [Davis, J. H., Nichol, C. P., Weeks, G., & Bloom, M. (1979) Biochemistry 18, 2103]. The orientational order in the fluid phase of both membranes decreased gradually with increasing temperature and was greater in the outer membrane than in the cytoplasmic membrane. The orientational order of the gel-phase component was the same for both membranes, within an experimental uncertainty of 10%, and was independent of temperature from 0 to 30 degrees C for the outer membrane and from 10 to 30 degrees C for the cytoplasmic membrane.
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The cytoplasmic and outer membranes of Escherichia coli were studied between 0 and 40 degrees C by deuterium magnetic resonance quadrupolar echo spectroscopy. The L51 strain of E. coli was used to incorporate perdeuterated palmitic acid into the membrane phospholipids. The cytoplasmic and outer membranes were separated using standard techniques. The spectrum of each membrane preparation was dominated at high temperatures (greater than or equal to 37 degrees C) by the characteristic liquid-crystalline plateau previously observed for perdeuterated palmitate chains in model phospholipid membranes. At low temperatures, the shape and width of the spectrum were characteristic of the gel phase. The relative intensities of the liquid-crystalline and gel features varied systematically with temperature. A quantitative analysis of the acyl chain orientational order was carried out by using the method of moments. The orientational order at each temperature was greater in the outer membrane sample than in that of the cytoplasmic membrane, indicating that the liquid-crystalline-gel transition region in the outer membrane is shifted to higher temperatures than that of the cytoplasmic membrane by about 7 degrees C. It is clear from the results that most of the phospholipid molecules participate in the phase transition.