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J Zajicek

Publications and source records attributed to J Zajicek.

At least 19 recordsLinked to original sources
PubMed2000

Sequence requirements for the N-methyl-D-aspartate receptor antagonist activity of conantokin-R.

Conantokin-R (con-R), a gamma-carboxyglutamate-containing 27-residue peptide, is a natural peptide inhibitor of the N-methyl-d-aspartate (NMDA) subtype glutamate receptor. Synthetic analogs of con-R were generated to evaluate the importance of the individual structural elements of this peptide in its NMDA receptor antagonist activity, measured by inhibition of the spermine-enhanced binding of the NMDA receptor-specific channel blocker, [(3)H]MK-801, to rat brain membranes. Progressive C-terminal truncations of the 27-residue peptide revealed stages of severe activity loss. These occurred at con-R[1-11] and con-R[1-7], corresponding to the deletions of Leu(12)-Pro(27) and Met(8)-Pro(27) respectively. A second set of analogs featured single Ala substitutions in the fully active con-R[1-17] fragment. The replacement of Met(8) and Leu(12) by Ala resulted in approximate 20- and 55-fold decreases of inhibitor potency, respectively. In addition to these two residues, the only other positions where a single Ala substitution led to substantial losses (from 11-fold to >1000-fold) of activity were those of the first five N-terminal amino acids. Based on the above findings, the binding epitope of con-R was localized to the N-terminal turn of the helix and other residues on one face along two subsequent turns. This contribution pattern of the side chains in activity closely resembles the results obtained with another member of this peptide family, conantokin-T. The secondary structure and metal ion binding properties of the con-R variants were also evaluated using circular dichroism spectroscopy. Divalent cation-dependent increases of alpha-helix content were observed in most analogs. However, analogs with replacement of Gla(11) and Gla(15), as well as truncation fragments shorter than 15 residues, lost the ability to be stabilized by metal ions. These results confirmed the location of the primary divalent cation binding locus at Gla(11) and Gla(15). Additional interactions were indicated by the reduced alpha-helix stability in the Ala analogs of Gla(4), Lys(7), and Arg(14).

1-Carboxyglutamic Acid↗
PubMed2000

The effects of ligand binding on the backbone dynamics of the kringle 1 domain of human plasminogen.

The internal motions of the backbone nitrogen atoms of the kringle 1 domain of human plasminogen (K1(Pg)) were examined in the absence and presence of the ligand, epsilon-aminocaproic acid. These dynamic properties were determined from (15)N NMR relaxation data in terms of the extended model-free parameters. The model of isotropic reorientation was found sufficient to account for overall molecular tumbling for both apo and EACA-bound K1(Pg). The global rotational correlation time (tau(m)) for apo-K1(Pg) was 5.87(+/-0.01) ns, while the tau(m) for ligand-bound K1(Pg) was 5.20(+/-0.01) ns, suggesting that perhaps some small degree of aggregation occurred in the apo form of the kringle module. Complexation of K1(Pg) with ligand mainly reduced those internal motions that occurred on a 100 ps to 5 ns time-scale. The magnitude of the chemical exchange was also attenuated upon ligand binding. These data are consistent with studies employing other approaches that suggest that the binding pocket is preformed in K1(Pg).

Aminocaproic Acid↗
PubMed2000

3-Hydroxy-3-methylglutaryl-CoA synthase. A role for glutamate 95 in general acid/base catalysis of C-C bond formation.

Replacement of 3-hydroxy-3-methylglutaryl-CoA synthase's glutamate 95 with alanine diminishes catalytic activity by over 5 orders of magnitude. The structural integrity of E95A enzyme is suggested by the observation that this protein contains a full complement of acyl-CoA binding sites, as indicated by binding studies using a spin-labeled acyl-CoA. Active site integrity is also demonstrated by (13)C NMR studies, which indicate that E95A forms an acetyl-S-enzyme reaction intermediate with the same distinctive spectroscopic characteristics measured using wild type enzyme. The initial reaction steps are not disrupted in E95A, which exhibits normal levels of Michaelis complex and acetyl-S-enzyme intermediate. Likewise, E95A is not impaired in catalysis of the terminal reaction step, as indicated by efficient catalysis of a hydrolysis partial reaction. Single turnover experiments indicate defective C-C bond formation. The mechanism-based inhibitor, 3-chloropropionyl-CoA, efficiently alkylates E95A. This is compatible with the presence of a functional general base, raising the possibility that Glu(95) functions as a general acid. Demonstration of a significant upfield shift for the methyl protons of HMG-CoA synthase's acetyl-S-enzyme reaction intermediate suggests a hydrophobic active site environment that could elevate the pK(a) of Glu(95) as required to support its function as a general acid.

Acyl Coenzyme A↗
PubMed2000

Deuterium nuclear spin-lattice relaxation times and quadrupolar coupling constants in isotopically labeled saccharides.

(13)C and (2)H spin-lattice relaxation times have been determined by inversion recovery in a range of site-specific (13)C- and (2)H-labeled saccharides under identical solution conditions, and the data were used to calculate deuterium nuclear quadrupolar coupling constants ((2)H NQCC) at specific sites within cyclic and acyclic forms in solution. (13)C T(1) values ranged from approximately 0.6 to 8.2 s, and (2)H T(1) values ranged from approximately 79 to 450 ms, depending on molecular structure (0.4 M sugar in 5 mM EDTA (disodium salt) in (2)H(2)O-depleted H(2)O, pH 4. 8, 30 degrees C). In addition to providing new information on (13)C and (2)H relaxation behavior of saccharides in solution, the resulting (2)H1 NQCC values reveal a dependency on anomeric configuration within aldopyranose rings, whereas (2)H NQCC values at other ring sites appear less sensitive to configuration at C1. In contrast, (2)H NQCC values at both anomeric and nonanomeric sites within aldofuranose rings appear to be influenced by anomeric configuration. These experimental observations were confirmed by density functional theory (DFT) calculations of (2)H NQCC values in model aldopyranosyl and aldofuranosyl rings.

Deuterium↗
PubMed1999

Comparison of C1q-receptors on rat microglia and peritoneal macrophages.

A comparison of the expression and ligand specificity of the C1q (first complement component) receptor on rat microglia and peritoneal macrophages was made. This revealed that radiolabelled C1q was competed from the peritoneal macrophages with intact C1q, and additively displaced by calf-skin collagen and purified C1q globular heads, suggesting the presence of at least two receptors. This was in contrast to microglia, where radiolabelled C1q was displaced with intact C1q and to a modest degree with collagen, but not with globular heads. Taken together, this implies that under these conditions, peritoneal macrophages and microglia both express a C1q receptor which binds to the collagen-like region, and that peritoneal macrophages additionally express a molecule which binds to the globular head of C1q. Analysis of the ligand bound by these cells reflected the differences observed in the competitive binding experiments, with the novel identification of naturally-occurring peptides from the globular head of C1q bound to the peritoneal macrophages, but not the microglia.

Amino Acid Sequence↗
PubMed1997

Metal-ion-binding properties of synthetic conantokin-G.

The secondary structure of the synthetic 17-residue peptide, conantokin-G (con-G), a gamma-carboxyglutamate-containing marine cone snail neuroactive protein, is altered from a random conformation to one containing a very high level (>70%) of alpha-helix on binding of multivalent cations. The proportion of alpha-helix formed correlated well with the size of the cation and ranged from a low of approx. 7% with large cations, such as Ba2+, to more than 70% with smaller cations, such as Mn2+, Mg2+ and Zn2+. The valency of the multivalent cation was not as important, since tervalent lanthanides (Eu3+, Gd3+ and Tb3+) of ionic radius 106-109 pm induced similar levels (50-60%) of helix to those induced by Ca2+ and Cd2+ (ionic radii 109 and 114 pm respectively). Although the correlation was not as tight, smaller cations of the same valency allowed the helical transition to occur at lower concentrations than the larger cations. The spectroscopic and spectrometric properties of some of these cations permitted a more detailed analysis of the molecular nature of the cation-con-G binding. EPR-based titrations with Mn2+ provided a binding isotherm that was deconvoluted to a single class of 2-3 Mn2+ sites of average Kd 3.9 microM. This number of sites was similar to that for Ca2+ [Prorok, Warder, Blandl and Castellino (1996) Biochemistry 35, 16528-16534], but a much lower Kd was displayed with Mn2+. Determinations by 1H NMR of the longitudinal relaxation rates of the water protons in Mn2+/con-G solutions at different magnetic field strengths corresponding to the proton Langmuir frequencies of 24, 300 and 500 MHz permitted calculation of the hydration number of Mn2+ in the complex, which was found to be 1.0. This indicates that five of the six co-ordination sites of Mn2+ are occupied by peptide atoms, probably oxygens. Titrations of the changes in Tb3+ fluorescence as a result of its binding to con-G gave an EC50 of 58 microM, a value nearly identical with that obtained by titration of the change in helicity of the peptide as a function of Tb3+ concentration. This shows that the macroscopic binding of Tb3+ to con-G is directly responsible for the alteration in secondary structure of the peptide. Finally, Cd2+ was found to be an extremely suitable cation for an NMR-based investigation of the amino acid residues of apo-con-G that are perturbed by cation binding. In a limited example of the results of this study, it was discovered that originally equivalent CH2(delta) protons of Arg13 became distinctly magnetically non-equivalent in the Cd2+-bound helical form of con-G. This indicates that Arg13 is situated in the helix in such a way that the mobility of its side chain is highly restricted. In conclusion, the data show that a variety of multivalent cations with measurable spectroscopic and spectrometric properties interact similarly with con-G and generate extensive alpha-helical conformation in this peptide.

Amino Acid Sequence↗
PubMed1997

Role of tryptophan-63 of the kringle 2 domain of tissue-type plasminogen activator in its thermal stability, folding, and ligand binding properties.

Conservative (F and Y) and radical (H and S) mutations have been engineered at a rigidly conserved aromatic residue, W63, of the isolated recombinant kringle 2 domain of tissue-type plasminogen activator (r-K2tPA), an amino acid residue predicted from the X-ray crystal structure to be important in the ligand binding properties of this isolated protein domain. The variants were expressed in Pichia pastoris cells. The binding constants of epsilon-aminocaproic acid (EACA), 7-aminoheptanoic acid (7-AHpA), and trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) to each of these mutant polypeptides were determined by titrations of the alterations in intrinsic fluorescence of the variant kringles with the ligands. As compared to wild-type r-K2tPA, increases in the Kd (dissociation) values of approximately 15-fold and 20-200-fold were found for the W63F and W63Y mutants, respectively, toward these three ligands. Neither the W63H nor the W63S variant interacted with these same ligands. Differential scanning calorimetric analyses were also performed on each of the peptides to determine whether the alterations affected the conformational stability of wtr-K2tPA. The data demonstrated that all of these mutants were thermally destabilized, possessing temperatures of maximum heat capacity (Tm) values that were 12-20 degrees C lower than that of wtr-K2tPA. Addition of EACA resulted in increases (approximately 12 degrees C) in the Tm values of r-[W63F]-K2tPA and r-[W63Y]K2tPA, a result showing that EACA stabilized the native conformations adopted by these kringle domains. As expected from its greatly diminished binding to r-[W63H]K2tPA and r-[W63S]-K2tPA, high concentrations of EACA had little effect on the Tm of thermal denaturation of these latter mutants. 1H-NMR analysis of the two aromatic mutant kringles was employed to assess their overall comparative folding properties. The high upfield chemical shifts (-0.98 ppm) of the CH3(delta') protons of L47, a major signal of proper kringle folding, were slightly lowered to -0.83 to -0.86 ppm in the cases of all of the mutants. This is due to alterations in the W25-L47 side-chain spatial orientations, possibly the result of slight conformational alterations that affect the distance relationships of these two amino acid side chains. Assignments of nearly all of the protons of the aromatic residues in the W63F and W63Y mutants were accomplished, and few additional differences from their wild-type counterpart were noted. Reactivities of the mutants against four different monoclonal antibodies directed to wtr-K2tPA revealed the possibility that some small local conformational alterations might have resulted from the residues that have replaced the W63. We conclude that W63 possesses an important direct role in the ligand binding properties of r-K2tPA. This residue also contributes significantly to the stability of the native conformation of this kringle domain and perhaps to maintenance of local conformations.

Cell Line↗
PubMed1997

Glial lineages and myelination in the central nervous system.

Oligodendrocytes, derived from stem cell precursors which arise in subventricular zones of the developing central nervous system, have as their specialist role the synthesis and maintenance of myelin. Astrocytes contribute to the cellular architecture of the central nervous system and act as a source of growth factors and cytokines; microglia are bone-marrow derived macrophages which function as primary immunocompetent cells in the central nervous system. Myelination depends on the establishment of stable relationships between each differentiated oligodendrocyte and short segments of several neighbouring axons. There is growing evidence, especially from studies of glial cell implantation, that oligodendrocyte precursors persist in the adult nervous system and provide a limited capacity for the restoration of structure and function in myelinated pathways damaged by injury or disease.

Adult↗
PubMed1995

Cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene is the committed step of taxol biosynthesis in Pacific yew.

The biosynthesis of taxol (paclitaxel) and related taxoids in Pacific yew (Taxus brevifolia) is thought to involve the cyclization of geranylgeranyl diphosphate to a taxadiene followed by extensive oxygenation of this diterpene olefin intermediate. A cell-free preparation from sapling yew stems catalyzed the conversion of [1-3H]geranylgeranyl diphosphate to a cyclic diterpene olefin that, when incubated with stem sections, was converted in good radiochemical yield to several highly functionalized taxanes, including 10-deacetyl baccatin III and taxol itself. Addition of the labeled olefin to a yew bark extract, followed by radiochemically guided fractionation, provided sufficient product to establish the structure as taxa-4(5),11(12)-diene by two-dimensional NMR spectroscopic methods. Therefore, the first dedicated step in taxol biosynthesis is the conversion of the universal diterpenoid precursor geranylgeranyl diphosphate to taxa-4(5),11(12)-diene, rather than to the 4(20),11(12)-diene isomer previously suggested on the basis of the abundance of taxoids with double bonds in these positions. The very common occurrence of taxane derivatives bearing the 4(20)-ene-5-oxy functional grouping, and the lack of oxygenated derivatives bearing a 4(5)-double bond, suggest that hydroxylation at C-5 of taxadiene with allylic rearrangement of the double bond is an early step in the conversion of this olefin intermediate to taxol.

Alkenes↗
PubMed1995

Hydroxycinnamic acid-derived polymers constitute the polyaromatic domain of suberin.

Suberin is an abundant, complex, intractable, plant cell wall polymeric network that forms both protective and wound-healing layers. Its function is, therefore, critical to the survival of all vascular plants. Its chemical structure and biosynthesis are poorly defined, although it is known to consist of both aromatic and aliphatic domains. While the composition of the aliphatic component has been fairly well characterized, that of the phenolic component has not. Using a combination of specific carbon-13 labeling techniques, and in situ solid state 13C NMR spectroscopic analysis, we now provide the first direct evidence for the nature of the phenolic domain of suberin and report here that it is almost exclusively comprised of a covalently linked, hydroxycinnamic acid-derived polymeric matrix.

Coumaric Acids↗
PubMed1995

1H and 13C NMR assignments and molecular modelling of a minor groove DNA-binding peptide from the HMG-I protein.

The HMG-I subfamily of high mobility group (HMG) chromatin proteins consists of DNA-binding proteins that preferentially bind to stretches of A.T-rich sequence both in vitro and in vivo. Recently, members of the HMG-I family have been suggested to bind in vitro to the narrow minor groove of A.T-DNA by means of an 11 amino acid peptide binding domain (BD) which, because of its predicted structure, is called the 'A.T-hook motif' [Reeves, R. & Nissen, M. (1990) J. Biol. Chem. 265, 8573-8582], and would appear to be crescent-shaped. A BD peptide with 13 amino-acid residues was synthesized and examined by proton and carbon-13 nuclear magnetic resonance (NMR) spectroscopy. The peptide contains four proline residues, and on the basis of NOEs and 13C chemical shifts was found to exist in an all-trans conformation. Molecular modelling based on this result provides evidence for a dynamic equilibrium between turn-like conformations in solution, the most populated of which is likely to be an S-shaped conformer, on the basis of amide exchange data.

Amino Acid Sequence↗
PubMed1995

Mechanisms of damage and repair in multiple sclerosis--a review.

Pathological features of MS include perivascular inflammation and demyelination with oligodendrocyte loss; in addition, attempts at remyelination are often unsuccessful and may culminate in astrocytic scarring. One approach to investigating the biological principles underlying these processes is to use in vitro systems to analyse single-cell behaviour as well as cell-cell interactions. This paper reviews such data concerned with cell injury and repair which illuminate both demyelination and remyelination. In tissue culture oligodendrocytes are susceptible to injury via cell-mediated and humoral mechanisms. Substances including complement and tumour necrosis factor are capable of killing rat oligodendrocytes in vitro; surface complement activation also initiates a number of intracellular processes within oligodendrocytes as well as providing ligands for phagocytic interactions. The reasons for oligodendrocyte complement activation are discussed, but it appears that species differences exist when extrapolating these data to humans. Myelination and remyelination can also be studied both in vitro and in vivo using defined cell populations. Results from these studies may eventually help to explain some pathological features of MS, including astrocytosis and factors governing the limits of remyelination.

Animals↗
PubMed1995

Human oligodendrocytes are not sensitive to complement. A study of CD59 expression in the human central nervous system.

BACKGROUND: One or more components of the oligodendrocyte-myelin unit are the target of immune attack in multiple sclerosis. The role of complement in this process has been suggested by the demonstration in vitro that rat oligodendrocytes are sensitive to lysis by Ab-independent complement attack, partly because of a lack of the complement regulatory protein molecule, CD59. EXPERIMENTAL DESIGN: This study assessed the sensitivity in vitro of human oligodendrocytes derived from neurosurgical specimens to complement attack and analyzed CD59 expression on their surface. The presence of CD59 was also examined in the human central nervous system during myelination and in both the normal and diseased adult brain. RESULTS: Human oligodendrocytes are insensitive in vitro to complement attack in the absence of Ab and using the Ab YTH 53.1 and were shown to possess CD59 on their surface. CD59 is absent from the human central nervous system before myelination, at which stage strong expression occurs in areas of myelin production. CD59 expression is then normally down-regulated but is particularly strong in reactive astrocytes in diseases such as multiple sclerosis. CONCLUSIONS: The findings suggest that the previous demonstration of rat oligodendrocyte complement sensitivity and lack of CD59 expression do not extend to the human central nervous system. There may be a role for CD59 in normal human myelination.

Adult↗
PubMed1994

Myelination in vitro of rodent dorsal root ganglia by glial progenitor cells.

Oligodendrocytes synthesize myelin in the mammalian central nervous system; they develop from glial progenitors which, at least in vitro, are bipotential and also differentiate into astrocytes. Maturation of these O-2A progenitors is known to be influenced by growth factors and by extracellular matrix molecules. We investigated the effect of neurons on glial development by co-culturing highly purified rodent embryonic dorsal root ganglia with neonatal O-2A progenitors. Neurons produce signals, including platelet-derived growth factor BB and basic fibroblast growth factor, which stimulate progenitor cells to synthesize DNA; axonal contact is associated with down-regulation in the expression of complex ganglioside surface molecules on O-2A progenitors; with maturation, many of these cells develop into oligodendrocytes allowing the normal process of myelination to take place, but neurons also promote the differentiation of type 2 astrocytes. This orchestration of proliferation and differentiation in O-2A progenitor cells favours the development of glial-neuronal interactions needed for saltatory conduction of the nerve impulse.

Animals↗
PubMed1992

Mechanisms of oligodendrocyte interaction with normal human serum--defining the role of complement.

The interaction of human serum with oligodendroglia was investigated in vitro using purified cultured neonatal rat oligodendrocytes. Previous evidence for antibody independent classical pathway complement activation was confirmed; the results also showed a deficit in the protection of rat oligodendrocytes from complement attack suggesting a deficiency in the expression of terminal regulatory proteins of the complement cascade. Thus, rat oligodendrocytes are selectively sensitive to normal serum due both to complement activation and impaired protection from terminal complement attack.

Animals↗