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Biomedical subjects

L Makowski

Publications and source records attributed to L Makowski.

At least 19 recordsLinked to original sources
PubMed2000

Lipoprotein profiles, not anthropometric measures, correlate with serum lipoprotein(a) values in children: the Taipei children heart study.

OBJECTIVE: Plasma lipoprotein(a) [Lp(a)] is a risk factor for cardiovascular disease. The purpose of this study is to evaluate the correlation of anthropometric measures, lipids and lipoprotein profiles and serum Lp(a) values among children in Taiwan. We will attempt to find parameters that will be able to predict Lp(a) levels in children. DESIGN AND METHODS: After a probability-proportional-to size, multi-stages sampling procedure, we randomly sampled 1500 schoolchildren from 10 schools in Taipei city. Anthropometric measures including body weight, body height, waist and hip circumference and skinfolds were measured. We used standard methods to measure serum total cholesterol (CHOL), triglycerides (TG), high density lipoprotein-cholesterol (HDL-C), apolipoprotein A1 and B (ApoA1 and ApoB) and Lp(a) levels. We also calculated low density lipoprotein-cholesterol (LDL-C) and CHOL HDL-C ratio (TCHR) by formula. RESULTS: We sampled 1283 children (635 boys and 648 girls) with a mean age of 13.3 years (from 12 to 16 years) in this study. The mean and medium serum Lp(a) levels were 16.8 and 8.8 mg/dl among boys and 20.8 and 11.9 mg/dl among girls. Children in the highest quintile of Lp(a) (mean = 49.6 and 58.6 mg/dl for boys and girls, respectively) had higher CHOL, LDL-C, ApoB levels and TCHR than children in the lowest quintile (mean = 3.1 and 3.7 mg/dl for boys and girls, respectively). Lipids and lipoprotein profiles, such as CHOL, LDL-C, Apo-B and TCHR were positively correlated with Lp(a) levels in both genders. Furthermore, the children with Lp(a) levels greater than or equal to 30 mg/dl had higher CHOL, LDL-C and Apo-B levels when compared to children with Lp(a) levels less than 30 mg/dl. After adjusting for age, cigarette smoking, alcohol drinking, puberty development and heart rates, LDL-C and ApoB levels were significantly positively associated with Lp(a) levels while ApoA1 was negatively associated among boys. Among girls, only Apo-B was significantly positively associated with Lp(a) and TG was negatively associated with Lp(a) levels. Most importantly, none of the anthropometric measures were significantly correlated with Lp(a) levels. CONCLUSIONS: From this study, we found that lipids and lipoproteins profiles, rather than degree of adiposity as reflected by anthropometric measures, are significantly associated with serum Lp(a) levels among school children.

Adolescent↗
PubMed2000

The likelihood function in fiber diffraction

The likelihood function is an appropriate target function for refinement of molecular structures using fiber diffraction data. However, its practical application to fiber diffraction faces two significant obstacles: (i) the intensities of layer lines in a fiber diffraction pattern usually arise from the superposition of several terms, each equivalent to a crystallographic structure factor, thereby making the calculation significantly more complex than for the crystallographic case; (ii) to describe a molecular structure at the atomic level based on fiber diffraction data, the radial and phase parts of the atomic coordinates must be treated separately owing to the uniaxial symmetry of the structure. These issues are addressed here in order to derive equations of likelihood functions for fiber diffraction. The special case of a single term on a layer line is treated first followed by extension of the method to the multiterm case. A practical difficulty in implementation of likelihood for the multiterm case is that each term has a different variance. An analytical technique is described that allows the conversion of the unequal-variance case to an equal-variance case. This makes it possible to express the likelihood by an explicit formula, allowing a direct implementation of the likelihood calculation. A cylindrically symmetric model is proposed for error distribution of the atomic coordinates in a helical structure. Variances and offset coefficients of the contributing terms in the likelihood functions are expressed in terms of the variance of the atomic coordinates in the cylindrical reference system.

Journal Article↗
PubMed1999

Screening of a library of phage-displayed peptides identifies human bcl-2 as a taxol-binding protein.

A random library of phage displayed peptides was screened for binding to a biotinylated derivative of paclitaxel (Taxol). Affinity-selected peptides were analyzed for similarity to human proteins. There was no significant similarity between the paclitaxel-selected peptides and tubulin. However, a subset of the peptides was identified that exhibits significant similarity to a non-conserved region of the anti-apoptotic human protein Bcl-2: ELISA assays confirmed binding of paclitaxel to Bcl-2, and circular dichroism spectroscopy demonstrated that a substantial conformational change accompanies this binding. In vivo, treatment with paclitaxel has been shown to lead to Bcl-2 inactivation with concomitant phosphorylation of residues in a disordered, regulatory loop region of the protein. Similarity between paclitaxel-selected peptides and this loop region implicate these residues in drug binding, and suggest that the apoptotic action of paclitaxel may involve the binding of paclitaxel to Bcl-2. These results demonstrate that peptides displayed on the surface of bacteriophage particles can mimic the ligand-binding properties of disordered regions of proteins.

Amino Acid Sequence↗
PubMed1999

Phage-display technology--finding a needle in a vast molecular haystack.

Screening of phage-displayed libraries of proteins and peptides has, for nearly a decade, proven to be a highly effective method for finding much needed 'needles' in a vast molecular 'haystack'. Over the past year, it has been used to solve an increasing diversity of problems, including identification of binding motifs for much smaller targets and the use of novel screening methods to identify chemical activities.

Bacteriophage M13↗
PubMed1999

Similarity between the sequences of taxol-selected peptides and the disordered loop of the anti-apoptotic protein, Bcl-2.

The anti-cancer drug taxol is known to bind to and induce the polymerization of tubulin and has recently been shown to bind to the anti-apoptotic protein Bcl-2, but not to its homolog, Bcl-XL. Libraries of random peptides displayed on the surface of a bacteriophage were screened to select those exhibiting affinity for taxol. The sequences of these peptides were compared to sequences of proteins involved in mitosis and apoptosis. No significant similarities were detected between the sequences of tubulins and the taxol-selected peptides. However, a high level of similarity exists between the selected peptides and the disordered loop of Bcl-2. Conversely, there was little similarity between the sequences of the selected peptides and Bcl-XL. These results indicate that peptides displayed on the surface of a bacteriophage can mimic the ligand-binding behavior of a disordered protein loop and that comparison of the sequences of affinity-selected peptides with protein sequences can be predictive for ligand binding.

Amino Acid Sequence↗
PubMed1999

Altered insulin secretion associated with reduced lipolytic efficiency in aP2-/- mice.

Recent studies have shown that genetic deficiency of the adipocyte fatty acid-binding protein (aP2) results in minor alterations of plasma lipids and adipocyte development but provides significant protection from dietary obesity-induced hyperinsulinemia and insulin resistance. To identify potential mechanisms responsible for this phenotype, we examined lipolysis and insulin secretion in aP2-/- mice. Beta-adrenergic stimulation resulted in a blunted rise of blood glycerol levels in aP2-/- compared with aP2+/+ mice, suggesting diminished lipolysis in aP2-/- adipocytes. Confirming this, primary adipocytes isolated from aP2-/- mice showed attenuated glycerol and free fatty acid (FFA) release in response to dibutyryl cAMP. The decreased lipolytic response seen in the aP2-/- mice was not associated with altered expression levels of hormone-sensitive lipase or perilipin. The acute insulin secretory response to beta-adrenergic stimulation was also profoundly suppressed in aP2-/- mice despite comparable total concentrations and only minor changes in the composition of systemic FFAs. To address whether levels of specific fatty acids are different in aP2-/- mice, the plasma FFA profile after beta-adrenergic stimulation was determined. Significant reduction in both stearic and cis-11-eicoseneic acids and an increase in palmitoleic acid were observed. The response of aP2-/- mice to other insulin secretagogues such as arginine and glyburide was similar to that of aP2+/+ mice, arguing against generally impaired function of pancreatic beta-cells. Finally, no aP2 expression was detected in isolated pancreatic islet cells. These results provide support for the existence of an adipo-pancreatic axis, the proper action of which relies on the presence of aP2. Consequently, aP2's role in the pathogenesis of type 2 diabetes might involve regulation of both hyperinsulinemia and insulin resistance through its impact on both lipolysis and insulin secretion.

Adipocytes↗
PubMed1998

Electron microscopy and x-ray diffraction studies of Lotus tetragonolobus A isolectin cross-linked with a divalent Lewisx oligosaccharide, an oncofetal antigen.

The interactions of lectins with multivalent carbohydrates often leads to the formation of highly ordered cross-linked lattices that are amenable to structural studies. A particularly well ordered, two-dimensional lattice is formed from fucose-specific isolectin A from Lotus tetragonolobus cross-linked with difucosyllacto-N-neohexaose, an oligosaccharide possessing the Lewisx determinant, which is an oncofetal antigen. A combination of electron microscopy, x-ray diffraction, simulation of electron micrographs, and molecular model building was used to determine the relative positions of the tetrameric lectin and bivalent carbohydrate within the lattice. X-ray diffraction from unoriented pellets was used to determine the lattice dimensions and analysis of electron micrographs was used to determine the lattice symmetry. Molecular models of the lattice were constructed based on the known structure of the jack bean lectin concanavalin A and the high degree of sequence homology between the two lectins. Using the symmetry and dimensions of the lattice and its appearance in filtered electron micrographs, molecular models were used to determine the orientation of the lectin in the lattice, and to define the range of lectin-oligosaccharide interactions consistent with the structural data. The present study provides the first description of a highly ordered, two-dimensional, cross-linked lattice between a tetravalent lectin and a bivalent carbohydrate.

Antigens, Neoplasm↗
PubMed1998

Bacterial adhesion pili are heterologous assemblies of similar subunits.

P-pili on uropathogenic bacteria are 68-A-diameter rods typically 1 microm in length. These structures project from the outer membrane of Escherichia coli, and contain on their distal tip a thin fibrillum, 25 A in diameter and 150 A long, displaying an adhesin protein responsible for the binding of the bacterium to the surface of epithelial cells lining the urinary tract. Operationally, it is possible to identify three morphologically distinct states of the 68-A-diameter P-pili rods, based on the degree of curvature each can adopt. These states are designated "straight," "curved," and "highly curved." The rods can also be unwound to form thin "threads" that are very similar to the tip fibrillae. Electron microscope data are used to distinguish among these four morphological states and to define limits on the shapes of the pilus proteins. The mechanical properties of the PapA polymers are assessed, and implications of rod polymorphism for pilus function are discussed. A wide variety of data are considered in light of the possibility that all pilins are similar in molecular architecture, with specific differences designed to optimize their specialized functions in the pilus assembly.

Amino Acid Sequence↗
PubMed1998

Analysis of the stability of hemoglobin S double strands.

The deoxyhemoglobin S (deoxy-HbS) double strand is the fundamental building block of both the crystals of deoxy-HbS and the physiologically relevant fibers present within sickle cells. To use the atomic-resolution detail of the hemoglobin-hemoglobin interaction known from the crystallography of HbS as a basis for understanding the interactions in the fibers, it is necessary to define precisely the relationship between the straight double strands in the crystal and the twisted, helical double strands in the fibers. The intermolecular contact conferring the stability of the double strand in both crystal and fiber is between the beta6 valine on one HbS molecule and residues near the EF corner of an adjacent molecule. Models for the helical double strands were constructed by a geometric transformation from crystal to fiber that preserves this critical interaction, minimizes distortion, and makes the transformation as smooth as possible. From these models, the energy of association was calculated over the range of all possible helical twists of the double strands and all possible distances of the double strands from the fiber axis. The calculated association energies reflect the fact that the axial interactions decrease as the distance between the double strand and the fiber axis increases, because of the increased length of the helical path taken by the double strand. The lateral interactions between HbS molecules in a double strand change relatively little between the crystal and possible helical double strands. If the twist of the fiber or the distance between the double strand and the fiber axis is too great, the lateral interaction is broken by intermolecular contacts in the region around the beta6 valine. Consequently, the geometry of the beta6 valine interaction and the residues surrounding it severely restricts the possible helical twist, radius, and handedness of helical aggregates constructed from the double strands. The limitations defined by this analysis establish the structural basis for the right-handed twist observed in HbS fibers and demonstrates that for a subunit twist of 8 degrees, the fiber diameter cannot be more than approximately 300 A, consistent with electron microscope observations. The energy of interaction among HbS molecules in a double strand is very slowly varying with helical pitch, explaining the variable pitch observed in HbS fibers. The analysis results in a model for the HbS double strand, for use in the analysis of interactions between double strands and for refinement of models of the HbS fibers against x-ray diffraction data.

Amino Acid Sequence↗
PubMed1997

X-ray diffraction from a helix of any length that displays cumulative azimuthal disorder.

An explicit formula has been derived to describe the attenuation and broadening of cylindrically averaged diffraction intensities from a helix of any given length which possesses cumulative azimuthal disorder. The application limits of an approximate formula, represented by the first term of this formula, are defined. Strategies to estimate the length of fibers, the degree of disorder, and the overlap of adjacent layer lines are outlined. Some features of diffraction patterns from the disordered helical structure of the HbS fiber are interpreted in light of these results. In these patterns, non-zero-order Bessel functions are attenuated and broadened due to azimuthal disorder and finite length. Adjacent layer lines overlap because of the very large axial repeat distance of the HbS fibers. As a result, the contribution of any Bessel function term with n > or = 10 is not discernible in these patterns. Only Bessel terms with n < 6 may be accurately estimated in these patterns, if instrumental broadening is negligible or correctable. The theory presented here may also be used to make a rough estimate of the degree of disorder in F-actin fibers by comparison of X-ray diffraction patterns with serial peak projections calculated assuming various degrees of disorder.

Actins↗
PubMed1997

Shuffling of structural elements in filamentous bacteriophages.

All class II filamentous bacteriophage coat proteins contain a conserved, 12-amino acid sequence highly homologous to the loop portion of the EF-hand Ca(2+)-binding motif. The Pf3 coat protein contains two regions of homology to this sequence. The 12-amino acid sequence corresponds to a region of the Pf1 coat protein whose structure is controversial. In some models of the virus structure, this region is alpha-helical. In others, it forms a loop that folds back on itself. The similarity of this region to the loop in the helix-loop-helix Ca(2+)-binding motif suggests that it takes on a loop structure in the virion. Each filamentous phage lacks at least one residue normally involved in Ca(2+)-coordination, consistent with the relatively weak Ca(2+)-binding properties of the filamentous phages. Consideration of the structure of the coat protein in the membrane and in the virus particle indicates that the protein may be more effective in binding cations in its membrane-bound form than in the virus particle. This suggests that release of cations from this loop may be an obligate step during assembly of the proteins into the virus particle.

Amino Acid Sequence↗
PubMed1996

Development of pilus organelle subassemblies in vitro depends on chaperone uncapping of a beta zipper.

The major subassemblies of virulence-associated P pili, the pilus rod (comprised of PapA) and tip fibrillum (comprised of PapE), were reconstituted from purified chaperone-subunit complexes in vitro. Subunits are held in assembly-competent conformations in chaperone-subunit complexes prior to their assembly into mature pili. The PapD chaperone binds, in part, to a conserved motif present at the C terminus of the subunits via a beta zippering interaction. Amino acid residues in this conserved motif were also found to be essential for subunit-subunit interactions necessary for the formation of pili, thus revealing a molecular mechanism whereby the PapD chaperone may prevent premature subunit-subunit interactions in the periplasm. Uncapping of the chaperone-protected C terminus of PapA and PapE was mimicked in vitro by freeze-thaw techniques and resulted in the formation of pilus rods and tip fibrillae, respectively. A mutation in the leading edge of the beta zipper of PapA produces pilus rods with an altered helical symmetry and azimuthal disorder. This change in the number of subunits per turn of the helix most likely reflects involvement of the leading edge of the beta zipper in forming a right-handed helical cylinder. Organelle development is a fundamental process in all living cells, and these studies shed new light on how immunoglobulin-like chaperones govern the formation of virulence-associated organelles in pathogenic bacteria.

Bacterial Proteins↗
PubMed1995

Methane-induced haemolysis of human erythrocytes.

Human erythrocytes were exposed to high concentrations of methane and nitrogen through the application of elevated partial pressures of these gas molecules. Cell leakage (haemolysis) was measured for cells exposed to these gases under a wide range of experimental conditions. Application of methane produces haemolysis at pressures far below the hydrostatic pressures known to disrupt membrane or protein structure. The effects of changes in buffer, temperature, diffusion rate and detergents were studied. Methane acts co-operatively with detergents to produce haemolysis at much lower detergent concentration than is required in the absence of methane or in the presence of nitrogen. At sufficiently high concentrations of methane, all cells are haemolysed. Increased temperature enhances the effect. Methane produces 50% haemolysis at a concentration of about 0.33 M compared with about 7.5 M methanol required for the same degree of haemolysis.

Buffers↗
PubMed1995

Structural polymorphism of bacterial adhesion pili.

Bacterial adhesion pili are designed to bind specifically and maintain attachment of bacteria to target cells. Uropathogenic P-pili are sufficiently mechanically resilient to resist the cleansing action of urine flow that removes most other bacteria. P-pili are 68 A in diameter and approximately 1 micron long, and are composed of approximately 1,000 copies of the principal structural protein, PapA. They are attached to the outer membrane by a minor structural protein, PapH and are terminated by an approximately 20 A diameter fibrillus composed of PapK, PapE and PapF, which presents the host-binding adhesin PapG. The amino-acid sequences of PapA, PapE, and PapF are similar, with highly conserved C-termini being responsible for binding to PapD, the periplasmic chaperone. Our three-dimensional reconstruction indicates that pili are formed by the tight winding of a much thinner structure. A structural transition allows the pilus to unravel without depolymerizing, producing a thin, extended structure five times the length of the original pilus.

Bacterial Adhesion↗