A nonlinear regression analysis of nonlinear, passive-deflation flow-volume plots.

When studying lung mechanics of intubated premature infants, by the passive-deflation technique we noted that in many flow-volume plots the descending limb was curvilinear with a convexity toward the volume axis. By conventional linear analysis lung mechanics of these patients did not change after the administration of terbutaline, but Mead's tangent-chord slope ratio method for quantifying the amount of curvature showed that the shape of the flow-volume plots did change. Because of the limitations of this method, we developed a microcomputer-based, reiterative regression algorithm which optimizes a nonlinear function for the best fit to any given set of data. We then studied six very low birth weight infants with clinical evidence of pulmonary gas trapping (weight at study, 1.22 +/- 0.29 kg; age, 26 +/- 16 days). We measured respiratory system resistance (Rrs), compliance (Crs), and expiratory time constants (TCrs) by the passive deflation technique before and after subcutaneous administration of 0.02 mg/kg of terbutaline. No effect of terbutaline in a dose sufficient to increase heart rate > 25 beats/min was observed. The same data analyzed using the nonlinear regression technique with a function based upon a two compartment model of parallel inhomogeneities revealed one compartment with relatively normal Rrs, Crs, and TCrs values, and a second compartment with a very high Rrs. The latter fell by 50% after terbutaline. These data suggest that abnormalities of airway resistance in ventilated preterm infants are not easily identifiable by classic linear analysis of lung mechanics.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the structure of dextran by 13C-nuclear magnetic resonance spectroscopy.

The 13C-n.m.r. spectra have been recorded for a series of dextrans whose structures, in terms of degree and type of branching, had previously been determined by methylation analysis. The spectra established that all observable linkages in these dextrans are alpha-linked. Correlation of the spectra with methylation data indicated that the 75-85-p.p.m. spectral region is diagnostic for establishing the presence of alpha-D-(1 leads to 2)-, alpha-D-(1 leads to 3)-, or alpha-D-(1 leads to 4)-linkages. Each chemical shift has been found to be temperature-dependent (deltadelta/deltaT) when referenced to either the deuterium lock or an external standard (tetramethylsilane). All carbohydrate deltadelta values are positive, and range from 0.01 to 0.03 p.p.m./degrees C. These values are considerably larger than analogous deltadelta/deltaT values previously observed for smaller molecules. Larger than average deltadelta/deltaT values are associated with the non-anomeric, sugar-linking carbon atoms.

Maternal lactoferrin in the urine of preterm infants. Evidence for retention of structure and function.

Intact (i.e., 78-kDa) lactoferrin has been purified from the urine of preterm infants fed human milk. The maternal origin of this lactoferrin, and the integrity of its primary structure have been documented. Computer analyses of the circular dichroism spectra revealed a composite secondary structure for the urinary lactoferrin that was indistinguishable from that of purified human milk lactoferrin and similar to that observed in the crystal structure. Intact function was suggested by iron binding; an approximate 2:1 molar ratio of iron to lactoferrin was confirmed. Thus, maternal lactoferrin is absorbed intact by the preterm infant and appears to remain structurally and functionally intact within the circulatory system and during urinary excretion. It is possible, therefore, that maternal lactoferrin has an immunoregulatory influence in newborn infants fed human milk.

Similarly I will not--cause abortion.

PIP: The writer opposes abortion in all cases, including rape, except to save the mother's life. "Liberalized" abortion laws place nonmedical responsibilities on doctors. Physicians should return to the Hippocratic Oath, which emphasizes their responsibilities to the community, their patients, and their profession. The Oath stresses the independent judgment of the doctor.^ieng

Abortion: a threat to the medical profession.

Changed attitudes toward abortion arouse interprofessional repercussions which threaten the medical profession. Inquiries in Europe revealed widespread physician opposition to abortion for non life-threatening indications, and deep divisions within the British medical profession as a result of legalization of such procedures in the U.K.The beginning of an individual's life reportedly remains uncertain despite current sophisticated instrumental techniques; therefore, hypothetical and actual life histories of a particular person are explored, illustrating that the demonstrations of Pasteur and of modern embryologists and geneticists amply verify medical tradition. Law, religion, and philosophy - however appropriately influencing physicians in other matters, and in augmenting their ethical stature - cannot, in the author's view, negate accurate observation or make proper that which tradition holds to be improper. At stake is our mutual trut, based on physicians sharing a fundamental regard for human life. If we lose that trust, we must know our consultants well, or chance placing our patients in jeopardy on account of nonmedical considerations.

Mechanism and rate of permeation of cells by polycyclic aromatic hydrocarbons.

The principal mechanism of cellular uptake of benzo(a)pyrene and other polycyclic aromatic hydrocarbons (PAH) from lipoproteins into cells is spontaneous transfer through the aqueous phase (Plant, A. L., Benson, D.M., and Smith, L.C. (1985) J. Cell Biol. 100, 1295-1308). Cellular uptake of benzo(a)pyrene from low density lipoproteins followed first-order kinetics with a rate constant that was independent of the relative lipoprotein concentrations or cell number but which was 2 orders of magnitude smaller than the rate constant for benzo(a)pyrene desorption from low density lipoproteins. Moreover, identical rate constants for cellular uptake of benzo(a)pyrene were observed when the donor vehicle was high density lipoproteins, very low density lipoproteins, or single bilayer phosphatidylcholine vesicles, even though rate constants for benzo(a)pyrene transfer from these donor vehicles differed by 10-fold. When phosphatidylcholine vesicles containing benzo(a)pyrene and a nontransferable fluorescence quencher were mixed with cells in a stopped-flow system, two kinetic components were distinguished: a fast component with a rate constant corresponding to that measured for transfer of benzo(a)pyrene out of vesicles, followed by a much slower component, with a time course approximating that measured for cellular accumulation of benzo(a)pyrene by other techniques. Rate constants for desorption of a series of PAH which contained different number of aromatic rings from phosphatidylcholine vesicles differed over a 70-fold range. First-order rate constants for cell uptake of benzo(a)pyrene and five other PAH of different molecular sizes had the same 70-fold range of values, but were 2 orders of magnitude smaller than their respective rate constants for desorption from single bilayer vesicles. In addition, activation energies for cell uptake were essentially identical to the respective activation energies for desorption of PAH from phosphatidylcholine vesicles, confirming the mechanistic similarity of the two processes.

Eigenanalysis of DAPI-stained chromosomes: tools and strategies toward computer-assisted analysis of FISH experiments.

The fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) is widely used as a chromosome counterstain in fluorescence in situ hybridization (FISH) studies. It produces a Q-banding pattern that allows for both chromosome identification and the assignment of molecular probes to specific chromosome bands. Using a statistical procedure based on eigenanalysis, we have extracted features from digital images of DAPI-stained chromosomes and constructed prototypes of each of the 24 human chromosomes. The features of these prototypes are directly proportional, in intensity profile and band location, to those of real chromosomes. The prototype's intensity profile can be translated into cytogenetic bands to provide a computer-based strategy for chromosome mapping and analysis amenable to automation. Data presented here were obtained using images from the 24 human chromosomes and mouse X chromosome. Moreover, the same procedure is general and can be used for the analysis of chromosomes from other species, as well as with banding techniques other than those using DAPI.

Apolipoproteins C-I, C-II, and C-III: kinetics of association with model membranes and intermembrane transfer.

The apoproteins (apo) C-I, C-II, and C-III are low molecular weight amphiphilic proteins that are associated with the lipid surface of the plasma chylomicron, very low density lipoprotein (VLDL), and high-density lipoprotein (HDL) subfractions. Purified apoC-I spontaneously reassociates with VLDL, HDL, and single-bilayer vesicles (SBV) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. ApoC-I also transfers reversibly from VLDL to HDL and from VLDL and HDL to SBV. The kinetics of association of the individual apoC proteins with SBV are second order overall and first order with respect to lipid and protein concentrations. At 37 degrees C, the rates of association were 2.5 x 10(10), 4.0 x 10(10) and 3.8 x 10(10) M-1 s-1 for apoC-I, apoC-II, and apoC-III, respectively. Arrhenius plots of association rate vs temperature were linear and yielded activation energies of 11.0 (apoC-I), 9.0 (apoC-II), and 10.6 kcal/mol (apoC-III). The kinetics of vesicle to vesicle apoprotein transfer are biexponential for intermembrane transfer, indicating two concurrent transfer processes. Rate constants at 37 degrees C for the fast component of dissociation were 11.7, 9.5, and 9.9 s-1, while rate constants for the slow component were 1.3, 0.6, and 0.9 s-1 for apoC-I, apoC-II, and apoC-III, respectively. The dissociation constants, Kd, of apoC-I, apoC-II, and apoC-III bound to the surface monolayer of phospholipid-coated latex beads were 0.5, 1.4, and 0.5 microM, respectively. These studies show that the apoC proteins are in dynamic equilibrium among phospholipid surfaces on a time scale that is rapid compared to lipolysis, lipid transfer, and lipoprotein turnover.

13C NMR studies of the thermal properties of a model high density lipoprotein. Apolipoprotein A-I-dimyristoylphosphatidylcholine complex.

The most abundant lipid and protein components of human plasma high density lipoproteins are phosphatidylcholine and apolipoprotein A-I (A-I). Under appropriate conditions, A-I spontaneously associates with dimyristoylphosphatidylcholine (DMPC) to quantitatively form a lipid-protein complex with a DMPC/A-I molar ratio of 100:1. Differential scanning calorimetry of this complex reveals two broad thermal transitions centered at approximately 27 and 72 degrees C. 13C NMR spectra of the complex have been obtained above, at, and below the lower transition temperature. The 13C resonance arising from the 3' carbon of the fatty acyl chains is a doublet, split by approximately 0.2 ppm, suggesting that the 3' carbon nuclei occupy two magnetically inequivalent sites. By replacing the sn-2 fatty acyl chain with myristate selectively 13C-enriched at carbon 3', we have shown that the splitting is, in fact, a result of magnetic inequivalence of the two sites and have assigned the lower field resonance to the 3' carbon nucleus of the sn-2 chain. The temperature dependence of the NMR relaxation rates indicates that the endothermic transition at 27 degrees C is associated with increased motional freedom for the phospholipids within this complex. The temperature dependence of the fatty acyl chain methylene 13C chemical shifts suggests that the population of gauche conformers increases above the transition temperature. These dynamic and conformational changes are characteristic of gel----liquid crystalline phase transitions observed in pure phospholipid systems. For the DMPC-A-I complex at 37 degrees C, the chemical shifts of the fatty acyl C 4'- 11' methylene envelope and of the C 7' and C 13' resonances occur significantly downfield from the corresponding chemical shifts for the DMPC vesicle. These results suggest that the apoprotein rigidifies the acyl chains by increasing their number of trans conformers.

The helical hydrophobic moment avoids prolines in phospholipid-binding proteins.

Proline residues appear to punctuate the distribution of amphiphilic phospholipid-binding regions of apolipoproteins. We have applied a quantitative test to this hypothesis and shown that the magnitude of the helical hydrophobic moment around proline residues is reduced in lipid-binding proteins and peptides.

Method for calculating 3-D coordinates from molecular stereograms.

The three-dimensional coordinates for the alpha-carbon atoms of crambin and basic pancreatic trypsin inhibitor (BPTI) were determined from the respective alpha-carbon trace stereograms using an improved Simplex algorithm. This algorithm was used in a two-step process to estimate the z-coordinate values. In one approach, an average interatomic distance value, an approximate viewing angle, and a table of digitized values for xleft, yleft and xright, yright are provided in the first step. In the second step, the z-coordinate values are derived by varying z to minimize the bond distance error (Rossmann and Argos, 1980). In another approach, only a reference bond distance table is provided along with the table of xleft, yleft and xright, yright digitized values. In the first step, the viewing angle (phi), a combined scale and viewing distance parameter (q), a rotational angular distortion from digitizing and/or photocopying (z), and translational distortion factors (xerr and yerr) are calculated. In the second step, the z-coordinate values are varied to minimize the bond distance error. RMS difference values of less than 1.5 A were obtained for both crambin and BPTI alpha-carbon atoms.

A structural assessment of the apo[a] protein of human lipoprotein[a].

Apolipoprotein[a], the highly glycosylated, hydrophilic apoprotein of lipoprotein[a] (Lp[a]), is generally considered to be a multimeric homologue of plasminogen, and to exhibit atherogenic/thrombogenic properties. The cDNA-inferred amino acid sequence of apo[a] indicates that apo[a], like plasminogen and some zymogens, is composed of a kringle domain and a serine protease domain. To gain insight into possible positive functions of Lp[a], we have examined the apo[a] primary structure by comparing its sequence with those of other proteins involved in coagulation and fibrinolysis, and its secondary structure by using a combination of structure prediction algorithms. The kringle domain encompasses 11 distinct types of repeating units, 9 of which contain 114 residues. These units, called kringles, are similar but not identical to each other or to PGK4. Each apo[a] kringle type was compared with kringles which have been shown to bind lysine and fibrin, and with bovine prothrombin kringle 1. Apo[a] kringles are linked by serine/threonine- and proline-rich stretches similar to regions in immunoglobulins, adhesion molecules, glycoprotein Ib-alpha subunit, and kininogen. In comparing the protease domains of apo[a] and plasmin, apo[a] contains a region between positions 4470 and 4492 where 8 substitutions, 9 deletions, and 1 insertion are apparent. Our analysis suggests that apo[a] kringle-type 10 has a high probability of binding to lysine in the same way as PGK4. In the only human apo[a] polymorph sequenced to date, position 4308 is occupied by serine, whereas the homologous position in plasmin is occupied by arginine and is an important site for proteolytic cleavage and activation. An alternative site for the proteolytic activation of human apo[a] is proposed.

Structure of an apolipoprotein-phospholipid complex: apoC-III induced changes in the physical properties of dimyristoylphosphatidylcholine.

The effect of ApoC-III, a major apoprotein constituent of human very low density lipoproteins, on the physical properties of dimyristoylphosphatidylcholine (DMPC) vesicles has been studied by magnetic resonance and fluorescence techniques. The sharp gel-liquid crystalline transition usually observed at 23 C in DMPC is both broadened and elevated when ApoC-III is bound as determined (a) from measurements of microscopic viscosity by pyrene excimer fluorescence, (b) from the distribution of di-tert-butyl nitroxide between the bulk aqueous phase and the fluid lipid phase, and (c) from the motion of fatty acyl chains of spin-labeled phosphatdylcholine. Experiments involving the translocation of ascorbate and charged nitroxide ions and the movement of paramagnetic Eu 3+ ions indicate that when ApoC-III binds to DMPC vesicles, it increases their permeability or destroys their original bilayer structure. These two possibilities were distinguishable by gel filtration of the DMPC-ApoC-III complex (approximately 34 mol mol) that indicated that the product particles were significantly smaller than the original vesicles. Taken together, the data indicate that ApoC-III binding to DMPC not only decreases the acyl chain motion of individual lipid molecules, but also induces break-down of bilamellar vesicular structure to give significantly smaller complexes.

Kinetics and mechanism of transfer of synthetic model apolipoproteins.

The effect of hydrophobicity on the rate and mechanism of transfer of a synthetic amphiphilic peptide between phosphatidylcholine single bilayer vesicles has been evaluated. These peptides, which had the sequence Cn-SSLKEYWSSLKESFS (where Cn represents a saturated acyl chain of n carbons that is attached to the amino terminus of the peptide and n = 8, 12, or 16), were distinguished by the length of the saturated acyl chain of n carbons that was covalently bonded to the amino terminus. The transfer of the peptides was monitored by following the rate of change of the intrinsic tryptophan fluorescence that followed mixing of donor vesicles, which contained peptide, phosphatidylcholine, and a fluorescence quencher, with acceptors composed only of phosphatidylcholine. The transfer rates were independent of the structure and concentration of the acceptor. The kinetics were biexponential with the contribution of the fast and slow components being nearly equal. The rates of both components decreased with increasing acyl chain length; the respective free energies of activation were linear with respect to the acyl chain length. These results showed that, unlike lipid transfer, peptide transfer is not always a simple unimolecular process. However, like lipid transfer, the transfer rates are a predictable function of hydrophobicity. It is proposed that the peptides exist as dimers on the phospholipid surface and that the two components of transfer are due to sequential transfer of each molecule in a dimer.

Structure and conformational analysis of lipid-associating peptides of apolipoprotein B-100 produced by trypsinolysis.

Apolipoprotein B-100 (apo B-100) contains putative lipid-associating regions that are, in part, responsible for its overall structure in human plasma low-density lipoproteins. Some of these regions have been identified by reassembly of the total tryptic peptides of apo B-100 with bovine brain sphingomyelin, 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and dimyristoylphosphatidylcholine (DPMC). Although more than 500 tryptic peptides are predicted from the known number of arginines and lysines in apo B-100, significant amounts of only 13 peptides spontaneously associate with all three phospholipids. These peptides share some structural characteristics, as predicted by several algorithms, that distinguish them from the water-soluble apolipoproteins. Most apolipoproteins associate with lipids via amphipathic helices and are highly helical in native and reassembled lipoproteins. Analysis of all apo B-100 lipophilic peptides by circular dichroism and by use of a predictive algorithm reveals no evidence of amphipathic helices. Although the predictive algorithm suggested that the lipophilic peptides of apo B-100 contain the sequence determinants for beta-sheet, no spectroscopic evidence for this structure was found. We conclude that the lipophilic regions of apo B-100 liberated by trypsinolysis are highly hydrophobic, although their secondary structures do not fit any simple model.

Magnetic resonance studies of apolipoprotein C-I nitroxide labeled or [13C]methyl enriched at methionine-38.

One of the three proposed lipid-binding regions of the human apolipoprotein C-I (apo-C-I) is an amphipathic helix which extends from residue 33 to residue 53 and includes a single methionine at sequence position 38. The involvement of the sequence around methionine-38 in phospholipid binding has been evaluated with paramagnetic and nuclear reported groups attached to the thiomethyl moiety. This moiety has been spin-labeled with N-(2,2,6,6-tetramethylpiperidinyl-1-oxy)bromoacetamide or 13C enriched with 13CH3I. As determined from its EPR spectrum, the nitroxide at Met-38 of apoC-I had a rotational correlation time (tau C) of 0.22 ns. When dimyristoylphosphatidylcholine (DMPC) was bound to the spin-labeled apoprotein, tau c increased to 0.35 ns, indicating decreased motion for the methionyl side chain. The line width (nu 1/2) and spin--lattice relaxation time (T1) for the thiomethyl resonance of 13C-enriched apoC-I in 10 mM phosphate buffer was 6.0 Hz and 320 ms, respectively. When the protein solution was made 1.6 M in Gdn-HCl, these values changed to 2.6 Hz and 970 ms, respectively. Upon addition of DMPC multilamellar liposomes to [13C]apoC-I in 1.6 M Gdn-HCl, the line width increased to 4.7 Hz and the T1 decreased to 380 ms. These results strongly suggest that methionine-38 of apoC-I resides in a region of the apoprotein which undergoes significant secondary and/or tertiary structural change upon disaggregation/unfolding in Gdn-HCl and upon interaction with phospholipid.

Biophysical properties of a major membrane phospholipid, dielaidoylphosphatidylethanolamine, found in an Escherichia coli fatty acid auxotroph.

Dielaidoylphosphatidylethanolamine, a principal lipid component of membranes of Escherichia coli fatty acid auxotrophs enriched in elaidic acid, has been studied by paramagnetic resonance, fluorescence, and calorimetric methods. EPR measurements with perdeutero-di-tert-butylnitroxide and 2,2,6,6-tetramethyl piperidine-1-oxyl indicate that, when dispersed in aqueous media, this phospholipid undergoes an abrupt order leads to disorder transition at 37.5 degrees C and 36.5 C, respectively. A similar transition temperature is suggested by experiments with 9-doxyl-dimyristoylphosphatidylethanolamine (DEPE). cis- and trans-Parinaric-acid fluorescence polarization measurements indicate that the midpoint of this transition occurs at 34.0 degrees C and 35.5 degrees C, respectively. Differential scanning calorimetry of DEPE revealed a single, sharp endotherm at 38.5 degrees C with increasing temperature; two exotherms of similar magnitude were observed at 36.5 degrees C and 34.5 degrees C upon cooling. This double transition was not observed by any of the other methods. From these results we conclude that the major structural transition at 30-31 degrees C observed previously with 5-, 12-, and 16-doxyl stearate in intact E. coli membranes is due to the DEPE present (Morrisett, J.D., Pownall, H.J., Plumlee, R.T., Smith, L.C., Zehner, Z.E., Esfahani, M., and Wakil, S.J. (1975) J. Biol. Chem. 250, 6969-6976).

Lecithin:cholesterol acyltransferase. Functional regions and a structural model of the enzyme.

The amino acid sequence of human lecithin:cholesterol acyltransferase has been determined by degradation and alignment of peptides obtained from tryptic and staphylococcal digestions and the cleavage with cyanogen bromide and consisted of 416 amino acid residues. All of the tryptic peptides of lecithin:cholesterol acyltransferase were isolated and sequenced. Peptides resulting from digestion by staphylococcal protease, cyanogen bromide cleavage, or the combination of the two methods were employed to find overlapping segments. The N terminus of human lecithin:cholesterol acyltransferase was determined to be phenylalanine by sequencing the whole protein up to 40 residues while the C terminus was identified as glutamic acid through carboxypeptidase Y cleavage. Cys50 and Cys74 and Cys313 and Cys356 were identified as the two disulfide bridges while the free sulfhydryl groups were located at positions 31 and 184. The N-glycosylated sites of the protein were assigned to asparagines at positions 20, 84, 272, and 384. The active site of lecithin:cholesterol acyltransferase was identified as serine on position 181 according to its homology with other serine-type esterases which have a common structure of glycine-variable amino acid-active serine-variable amino acid-glycine (Gly-X-Ser-X-Gly) with the variable amino acids disrupting the homology. No long internal repeats or homologies with apolipoproteins were found. The secondary structure is consistent with the results of predictive algorithms. A simple model of the enzyme is proposed on the basis of available chemical data and predictive methods.

Mapping segmental imbalances using comparative genomic hybridization and eigenanalysis.

We have tested a new approach to comparative genomic hybridization (CGH) analysis using digital ratio images and eigenanalysis, which allows the recognition of consistent patterns along the chromosomes and discards random (background noise) patterns. We have performed test experiments using genomic DNAs from a patient with a duplication, another with a deletion of a chromosome segment, and a prostate cancer biopsy. Image ratio analysis was performed, and ratio images of the relevant chromosome were subjected to eigenanalysis. The results showed a high-contrast enhancement of the regions corresponding to the unbalanced genomic segment, with clearly defined limits between normal and abnormal fluorescence ratios. The combination of digital ratio images and eigenanalysis allowed the precise mapping of unbalanced regions consistent with other methods of analysis. Because there is no limit to the number of chromosomes that can be analyzed at any one time, the method has the potential of increasing the sensitivity of CGH by reducing the noise component.