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S Knapp

Publications and source records attributed to S Knapp.

At least 37 records · Page 2Linked to original sources
PubMed2000

Crystallographic evidence for substrate-assisted catalysis in a bacterial beta-hexosaminidase.

beta-Hexosaminidase, a family 20 glycosyl hydrolase, catalyzes the removal of beta-1,4-linked N-acetylhexosamine residues from oligosaccharides and their conjugates. Heritable deficiency of this enzyme results in various forms of GalNAc-beta(1,4)-[N-acetylneuraminic acid (2,3)]-Gal-beta(1,4)-Glc-ceramide gangliosidosis, including Tay-Sachs disease. We have determined the x-ray crystal structure of a beta-hexosaminidase from Streptomyces plicatus to 2.2 A resolution (Protein Data Bank code ). beta-Hexosaminidases are believed to use a substrate-assisted catalytic mechanism that generates a cyclic oxazolinium ion intermediate. We have solved and refined a complex between the cyclic intermediate analogue N-acetylglucosamine-thiazoline and beta-hexosaminidase from S. plicatus to 2.1 A resolution (Protein Data Bank code ). Difference Fourier analysis revealed the pyranose ring of N-acetylglucosamine-thiazoline bound in the enzyme active site with a conformation close to that of a (4)C(1) chair. A tryptophan-lined hydrophobic pocket envelopes the thiazoline ring, protecting it from solvolysis at the iminium ion carbon. Within this pocket, Tyr(393) and Asp(313) appear important for positioning the 2-acetamido group of the substrate for nucleophilic attack at the anomeric center and for dispersing the positive charge distributed into the oxazolinium ring upon cyclization. This complex provides decisive structural evidence for substrate-assisted catalysis and the formation of a covalent, cyclic intermediate in family 20 beta-hexosaminidases.

Acetylglucosamine↗
PubMed2000

Synthesis of the sialidase inhibitor siastatin B.

[structure] The resolved piperidinecarboxylate (R)-7 was converted to siastatin B (1) by an efficient and stereoselective sequence that includes a bromo-beta-lactonization and an N-acyliminium azidation. Two analogues (3 and 4) of siastatin were also prepared.

Azides↗
PubMed2000

Aminohexanoic hydroxamate is a potent inducer of the differentiation of mouse neuroblastoma cells.

Deoxyhypusine synthase is the key enzyme for modifying a lysine residue to hypusine in the cellular protein eukaryotic initiation factor 5A (eIF-5A). Deletion of the deoxyhypusine synthase or the eIF-5A gene in yeast produces lethal phenotype. Inhibition of deoxyhypusine synthase by 1-guanidino-7-aminoheptane (GC7) suppresses tumor cell growth. Hypusine formation represents one of the most specific polyamine-dependent biochemical reactions. In view of the importance of polyamines in growth regulation and cancer biology, deoxyhypusine synthase has been considered to be a good target for chemotherapeutic drug design. Using GC7 as a prototype we have synthesized and tested three classes of diamine analogs, namely, guanidino-, pyrimidino-, and hydroxamate derivatives, as potential inhibitors for deoxyhypusine synthase. Our study shows that (i) among all the compounds tested, GC7 remained to be the most potent inhibitor for deoxyhypusine synthase; (ii) N,N'-bispyrimidino-1, 9-diaminononane, although a poor inhibitor of deoxyhypusine synthase, was a potent growth inhibitor; and (iii) one of the hydroxamate derivatives, 6-aminohexanoic hydroxamate (HC6), prominently induced the differentiation of mouse neuroblastoma cells at sub-millimolar concentrations. Interestingly, other hydroxamates with different chain length were not nearly as effective as HC6 in inducing neuroblastoma cell differentiation. The effect of HC6 was also unique in that it could induce neurite outgrowth and the expression of neuron-specific genes such as synapsin I and MAP-2 in neuroblastoma cells in the absence of other promoting agents such as cAMP. The effect of HC6 on neuroblastoma cell differentiation was comparable with, or better than that of N(6),O(2)'-dibutyryl cAMP (Bt(2)cAMP), a standard reagent commonly used for inducing the differentiation of mouse and human neuroblastoma cells in culture.

3T3 Cells↗
PubMed2000

Copper(II) and nickel(II) complexes of binucleating macrocyclic bis(disulfide)tetramine ligands.

Novel macrocyclic bis(disulfide)tetramine ligands and several Cu(II) and Ni(II) complexes of them with additional ligands have been synthesized by the oxidative coupling of linear tetradentate N2S2 tetramines with iodine. Facile demetalation of the Ni(II) oxidation products affords the free 20-membered macrocycles meso-9 and rac-9 and the 22-membered macrocycle 16, all of which are potentially octadentate N4S4 ligands. X-ray structure analyses reveal distinctly different conformations for the two isomers of 9; meso-9 shows a stepped conformation in profile with the disulfide groups corresponding to the rise of the step, whereas rac-9 exhibits a V conformation with the disulfide groups near the vertex of the V. No metal complexes of rac-9 have been isolated. Crystallographic studies of three Cu(II) complexes reveal that depending upon the size of the macrocyclic ligand and the nature of the additional ligands (I-, NCO-, and CH3CN), the Cu(II) coordination geometry shows considerable variation (plasticity), with substantial changes in the Cu(II)-disulfide bonding. Thus, a diiodide salt contains six-coordinate Cu(II) to which all four bridging disulfide sulfur atoms form strong equatorial bonds. In contrast, isocyanato complexes of the 20- and 22-membered macrocycles exhibit trigonal-bipyramidal Cu(II) and distorted cis-octahedral Cu(II) geometries, respectively, having only one and no short equatorially bound sulfur atoms. The coordination geometry of the latter complex can also be described as four-coordinate seesaw with two semicoordinated S(disulfide) ligands. Disulfide-->Cu(II) ligand-to-metal charge transfer absorptions of both isocyanato-containing Cu(II) species appear too weak to observe, probably because of poor overlap of the sulfur orbitals with the Cu(II) d-vacancy. The dual disulfide-bridged Ni(II) units of the crystallographically characterized octahedral Ni(II) complex of meso-9 with axial iodide and acetonitrile ligands promote substantial antiferromagnetic coupling (J = -13.0(2) cm-1).

Journal Article↗
PubMed2000

Synthesis of the ezomycin nucleoside disaccharide.

[equation--see text] A protected ezomycin octosyl nucleoside was glycosylated at O-6' with a protected ezoaminuroic acid donor to afford, following several functional group modifications, the title compound 1 ( identical with 4-desamino-4-oxoezomycin A(2)).

Aminoglycosides↗
PubMed2000

Risk management and life-threatening patient behaviors.

This article reviews the legal and ethical issues that arise in the treatment of patients who may pose a threat to harm themselves or others. The recent practice of developing empirically validated treatments has not yet been applied to the diagnosis and treatment of patients who present an imminent danger of harm to themselves or others. Instead, psychologists must follow standards that come primarily from court cases or statutes. Although these standards have some degree of commonality across North America, psychologists are encouraged to be informed about local laws and court cases.

Child↗
PubMed2000

Prostaglandin E(1) is able to increase migration of leukocytes through endothelial cell monolayers.

Leukocyte interactions with endothelial cells play an important role during inflammatory processes. Leukocytes pass a monolayer of endothelial cells (ECM) to migrate into the extravascular space. The aim of the current study was to investigate whether prostaglandin E(1) (PGE(1)) influences the process of leukocyte migration. In a prospective controlled study, the influence of prostaglandin E(1) (50-5000 ng/mL) on leukocyte migration through endothelial cell monolayers (n = 7) was investigated. Human umbilical endothelial cells (HUVEC) and/or leukocytes were preincubated with clinically relevant, higher, and lower concentrations of prostaglandin E(1) and the amount of leukocyte migration after 3 h was measured. HUVEC were cultured on microporous membrane filters until achievement of a monolayer for investigation of leukocyte migration. Polymorphonuclear leukocytes (PMNL) were isolated from healthy volunteers and PMNL migration was studied under the influence of PGE(1). In clinically relevant concentrations, PGE(1) was able to increase significantly leukocyte migration through endothelial cell monolayers (205 +/- 7.8%, P < 0.05 compared to control; when treating PMNL alone, migration rate was 120 +/- 9.2% compared to control, ns; only endothelial cell monolayers treated up to 145 +/- 10.2%, P < 0.05 compared to control) showing a dose-dependent effect. In this assay, both cell types (PMNL and ECM) could be treated simultaneously, simulating the clinical situation after an iv administration. In conclusion, PGE(1) is able to increase leukocyte migration through endothelial cell monolayers when both cell types are pretreated. The treatment of either leukocytes or endothelial cell monolayers in the cell coculture showed no significant increase. These findings support the theory that prostaglandins may play a major role during inflammation. Future clinical studies are warranted to confirm this hypothesis.

Adult↗
PubMed2000

Prostaglandin E(1) does not influence plasmatic coagulation, hepatic synthesis, or postoperative blood loss in patients after coronary-artery bypass grafting.

STUDY OBJECTIVE: To assess whether postoperatively administered prostaglandin E1 (PGE1) might prevent bleeding in patients after coronary artery bypass grafting (CABG). DESIGN: Prospective, randomized, placebo-controlled trial. SETTING: University-affiliated hospital. PATIENTS: 49 patients scheduled for elective CABG surgery. INTERVENTIONS: The PGE1 group received intravenous PGE(1) up to 15 ng/kg/min for 72 hours after surgery, whereas the placebo group received isotonic saline for the same time period. MEASUREMENTS AND MAIN RESULTS: Nine patients (4 in the PGE1 group vs. 5 in the placebo group) had to be excluded because of hemodynamic instability, and 1 in the placebo group because of gastric bleeding. In the remaining 39 patients (20 vs. 19), no significant differences with regard to hemoglobin levels or platelet count could be observed. There was no significant difference between the groups concerning the amount of packed red blood cells, platelet concentrates, or fresh frozen plasma transfused. No significant differences could be observed regarding laboratory markers of coagulation activation or hepatic synthesis either. CONCLUSIONS: PGE1 did not prevent coagulation disturbances and blood loss when administered postoperatively in patients undergoing CABG. The absence of these expected effects might be explained by the concomitant administration of acetylsalicylic acid, whose antiaggregatory acivity seems to exceed the effects of PGE1.

Aged↗
PubMed2000

Immunoadsorption in Goodpasture's syndrome.

Patients with Goodpasture's syndrome presenting with dialysis-dependent end-stage renal failure at diagnosis almost never regain independent renal function. We report a patient with a 100% crescentic lesion in whom reversal of dialysis dependence was achieved by immunoadsorption together with immunosuppression. In a second patient, early initiation of immunoadsorption was able to completely restore normal renal function as early as 1 month after the start of treatment. These data give evidence of the use of immunoadsorption as a hopeful alternative to conventional plasma exchange in patients with Goodpasture's syndrome showing advanced renal failure.

Adolescent↗
PubMed2000

Thermal unfolding and conformational stability of the recombinant domain II of glutamate dehydrogenase from the hyperthermophile Thermotoga maritima.

Domain II (residues 189-338, M(r) = 16 222) of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was used as a model system to study reversible unfolding thermodynamics of this hyperthermostable enzyme. The protein was produced in large quantities in E.COLI: using a T7 expression system. It was shown that the recombinant domain is monomeric in solution and that it comprises secondary structural elements similar to those observed in the crystal structure of the hexameric enzyme. The recombinant domain is thermostable and undergoes reversible and cooperative thermal unfolding in the pH range 5.90-8.00 with melting temperatures between 75.1 and 68.0 degrees C. Thermal unfolding of the protein was studied using differential scanning calorimetry and circular dichroism spectroscopy. Both methods yielded comparable values. The analysis revealed an unfolding enthalpy at 70 degrees C of 70.2 +/- 4.0 kcal/mol and a DeltaC(p) value of 1.4 +/- 0.3 kcal/mol K. Chemical unfolding of the recombinant domain resulted in m values of 3.36 +/- 0.10 kcal/mol M for unfolding in guanidinium chloride and 1.46 +/- 0.04 kcal/mol M in urea. The thermodynamic parameters for thermal and chemical unfolding equilibria indicate that domain II from T.MARITIMA: glutamate dehydrogenase is a thermostable protein with a DeltaG(max) of 3.70 kcal/mol. However, the thermal and chemical stabilities of the domain are lower than those of the hexameric protein, indicating that interdomain interactions must play a significant role in the stabilization of T. MARITIMA: domain II glutamate dehydrogenase.

Bacterial Proteins↗
PubMed2000

Outcome and prognostic factors in critically ill cancer patients admitted to the intensive care unit.

OBJECTIVE: To assess survival in cancer patients admitted to an intensive care unit (ICU) with respect to the nature of malignancy, cause of ICU admittance, and course during ICU stay as well as to evaluate the prognostic value of the Acute Physiology and Chronic Health Evaluation (APACHE) III score. DESIGN: Retrospective cohort study. SETTING: ICU at a university cancer referral center. PATIENTS: A total of 414 cancer patients admitted to the ICU during a period of 66 months. INTERVENTIONS: None. MEASUREMENTS: Charts of the patients were analyzed with respect to underlying disease, cause of admission, APACHE III score, need and duration of mechanical ventilation, neutropenia and development of septic shock, as well as ICU survival and survival after discharge. Mortality data were compared with two control groups: 1362 patients admitted to our ICU suffering from diseases other than cancer and 2,776 cancer patients not admitted to the ICU. MAIN RESULTS: ICU survival was 53%, and 1-yr survival was 23%. The 1-yr mortality rate was significantly lower in both control groups. Patients admitted after bone marrow transplantation had the highest mortality. In a multivariate analysis, prognosis was negatively influenced by respiratory insufficiency, the need of mechanical ventilation, and development of septic shock during the ICU stay. Admission after cardiopulmonary resuscitation yielded high ICU mortality but a relatively good long-term prognosis. Admission after surgery and as a result of acute hemorrhage was associated with a good prognosis. Age, neutropenia, and underlying disease did not influence outcome significantly. Admission APACHE III scores were significantly higher in nonsurvivors but failed to predict individual outcome satisfactorily. All patients with APACHE III scores of >80 died at the ICU. CONCLUSION: A combination of factors must be taken into account to estimate a critically ill cancer patient's prognosis in the ICU. The APACHE III scoring system alone should not be used to make decisions about therapy prolongation. Admission to the ICU worsens the prognosis of a cancer patient substantially; however, as ICU mortality is 47%, comparable with severely ill noncancer patients, general reluctance to admit cancer patients to an ICU does not seem to be justified.

APACHE↗
PubMed2000

A spiro-linked pyrene-naphthoquinone.

The title molecule, 2'-pyrenylspiro[2, 3-dihydro-1H-cyclopenta[b]naphthalene-2,5'-1',3'-dioxane]-4,9-dione, C(32)H(22)O(4), contains an electron-donating pyrene group spiro-linked to an electron-accepting naphthoquinone. The molecules are V-shaped in profile and stack to form columns along b with alternating, approximately coplanar, pyrene and naphthoquinone fragments. Intermolecular contacts within a column are consistent with some degree of pi contact and possible long-range delocalization. Individual columns form a herringbone pattern when the crystal is viewed along b.

Crystallography, X-Ray↗
PubMed1999

Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. II: construction of a 16-residue ion-pair network at the subunit interface.

The role of an 18-residue ion-pair network, that is present in the glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus, in conferring stability to other, less stable homologous enzymes, has been studied by introducing four new charged amino acid residues into the subunit interface of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima. These two GDHs are 55 % identical in amino acid sequence, differ greatly in thermo-activity and stability and derive from microbes with different phylogenetic positions. Amino acid substitutions were introduced as single mutations as well as in several combinations. Elucidation of the crystal structure of the quadruple mutant S128R/T158E/N117R/S160E T. maritima glutamate dehydrogenase showed that all anticipated ion-pairs are formed and that a 16-residue ion-pair network is present. Enlargement of existing networks by single amino acid substitutions unexpectedly resulted in a decrease in resistance towards thermal inactivation and thermal denaturation. However, combination of destabilizing single mutations in most cases restored stability, indicating the need for balanced charges at subunit interfaces and high cooperativity between the different members of the network. Combination of the three destabilizing mutations in triple mutant S128R/T158E/N117R resulted in an enzyme with a 30 minutes longer half-life of inactivation at 85 degrees C, a 3 degrees C higher temperature optimum for catalysis, and a 0.5 degrees C higher apparent melting temperature than that of wild-type glutamate dehydrogenase. These findings confirm the hypothesis that large ion-pair networks do indeed stabilize enzymes from hyperthermophilic organisms.

Amino Acid Substitution↗
PubMed1999

The staphylococcal alpha-toxin pore has a flexible conformation.

The alpha-toxin from Staphylococcus aureus undergoes several conformational changes from the time it is released from the bacterium to the moment it forms a channel in the plasma membrane of its target cell. It is initially a soluble monomer, which undergoes membrane binding and oligomerization into a heptameric ring and finally inserts into the lipid bilayer to form a pore. Here we have analyzed the stability of different forms of the alpha-toxin (monomer as well as heptamers in solution, bound to the membrane and membrane-inserted) by differential scanning calorimetry and limited proteolysis. Data presented here show that, in contrast to both the membrane-bound prepore complex and the monomer in solution, the membrane-inserted alpha-toxin channel does not undergo cooperative unfolding and is highly susceptible to proteases. These observations suggest that the channel has a looser conformation. Interestingly, resistance to proteases could be recovered upon solubilization of the channel, indicating that the loss of rigid tertiary packing only occurred upon membrane insertion. Far-UV CD data, however, suggest that the transmembrane beta-barrel must be stably folded and that therefore only the Cap and Rim domains of the channel are loosely packed. All together, our data show that the alpha-toxin channel is not a rigid complex within the membrane but adopts a rather flexible conformation.

Bacterial Toxins↗
PubMed1999

Enhancement of free fatty acid incorporation into phospholipids by choline plus cytidine.

Cytidine and choline, present in cytidine 5'-diphosphate choline (CDP-choline), are major precursors of the phosphatidylcholine found in cell membranes and important regulatory elements in phosphatide biosynthesis. Administration of CDP-choline to rats increases blood and brain cytidine and choline levels; this enhances the production of endogenous CDP-choline which then combines with fatty acids (as diacylglycerol), to yield phosphatidylcholine. We examined the effect of providing cytidine and choline on incorporation of free fatty acids into phosphatidylcholine and other major phospholipids in PC12 cells. Addition of equimolar cytidine and choline (100-500 microM) to [3H]-arachidonic acid (50 microM, 0.2 microCi, bound to bovine serum albumin) dose-dependently increased the accumulations of [3H]-phosphatidylcholine (PtdCho), [3H]-phosphatidylinositol (PtdIno) and [3H]-phosphatidylethanolamine (PtdEtn) (by up to 27+/-3%, 16+/-3% and 11+/-3%, respectively, means+/-S.E.M.). This effect was seen with 8-18 h of incubation. The incorporation of [3H]-oleic acid into [3H]-PtdCho was even more enhanced (by up to 42+/-3%) as were the incorporations of [14C]-choline and [3H]-glycerol. The effects of choline and cytidine were enhanced by 12-O-tetradecanoylphorbol-13-acetate (TPA, 1 microM), which activates CTP:phosphocholine cytidylyltransferase (CT) and facilitates choline uptake. Replacing choline by ethanolamine also enhanced the incorporation of [3H]-arachidonic acid into [3H]-PtdEtn, [3H]-PtdIno and [3H]-PtdCho. Arachidonic acid (10-200 microM) alone failed to affect the incorporation of [14C]-choline into phosphatidylcholine. We suggest that the increases in phospholipid synthesis caused by concurrent cytidine and choline supplementation enhance the incorporation of arachidonic acid and certain other fatty acids into the major glycerophospholipids. Removing these fatty acids as source of potentially toxic oxidation products could contribute to the beneficial effects of CDP-choline in treating stroke or other brain damage.

Animals↗
PubMed1999

Prostaglandin E2 stimulates amyloid precursor protein gene expression: inhibition by immunosuppressants.

Amyloid plaques that accumulate in the brains of patients with Alzheimer's disease (AD) are primarily composed of aggregates of amyloid peptides that are derived from the amyloid precursor protein (APP). Overexpression of APP in cell cultures increases the formation of amyloidogenic peptides and causes neurodegeneration and cognitive dysfunction in transgenic mice. We now report that activation of prostaglandin E2 (PGE2) receptors increases cAMP formation and stimulates overexpression of APP mRNA and holoprotein in primary cultures of cortical astrocytes. Levels of glial fibrillary acidic protein were also increased by PGE2 treatment, suggesting that these cultured astrocytes resemble reactive astrocytes found in vivo. The stimulation by PGE2 of APP synthesis was mimicked or blocked by activators or inhibitors, respectively, of protein kinase A. Actinomycin D or cycloheximide also inhibited the increase in APP holoprotein stimulated by PGE2. Treatment of astrocytes with 8-Bromo-cAMP or forskolin for 24 hr also stimulated APP overexpression in cultured astrocytes. The immunosuppressants cyclosporin A and FK-506 inhibited the increase in APP mRNA and holoprotein levels caused by PGE2 or by other treatments that elevated cellular cAMP levels; the inhibitory effect of FK-506 but not of cyclosporin A was attenuated by rapamycin. These results suggest that prostaglandins produced by brain injury or inflammation can activate APP transcription in astrocytes and that immunosuppressants may be used to prevent APP overexpression and possibly the pathophysiological processes underlying AD.

Amyloid beta-Protein Precursor↗