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Biomedical subjects

F X Zhou

Publications and source records attributed to F X Zhou.

At least 19 recordsLinked to original sources
PubMed2001

Polar residues drive association of polyleucine transmembrane helices.

Although many polar residues are directly involved in transmembrane protein functions, the extent to which they contribute to more general structural features is still unclear. Previous studies have demonstrated that asparagine residues can drive transmembrane helix association through interhelical hydrogen bonding [Choma, C., Gratkowski, H., Lear, J. D. & DeGrado, W. F. (2000) Nat. Struct. Biol. 7, 161-166; and Zhou, F. X., Cocco, M. J., Russ, W. P., Brunger, A. T. & Engelman, D. M. (2000) Nat. Struct. Biol. 7, 154-160]. We have studied the ability of other polar residues to promote helix association in detergent micelles and in biological membranes. Our results show that polyleucine sequences with Asn, Asp, Gln, Glu, and His, residues capable of being simultaneously hydrogen bond donors and acceptors, form homo- or heterooligomers. In contrast, polyleucine sequences with Ser, Thr, and Tyr do not associate more than the polyleucine sequence alone. The results therefore provide experimental evidence that interactions between polar residues in the helices of transmembrane proteins may serve to provide structural stability and oligomerization specificity. Furthermore, such interactions can allow structural flexibility required for the function of some membrane proteins.

Amino Acid Sequence↗
PubMed2000

Interhelical hydrogen bonding drives strong interactions in membrane proteins.

Polar residues in transmembrane alpha-helices may strongly influence the folding or association of integral membrane proteins. To test whether a motif that promotes helix association in a soluble protein could do the same within a membrane, we designed a model transmembrane helix based on the GCN4 leucine zipper. We found in both detergent micelles and biological membranes that helix association is driven strongly by asparagine, independent of the rest of the hydrophobic leucine and/or valine sequence. Hydrogen bonding between membrane helices gives stronger associations than the packing of surfaces in glycophorin A helices, creating an opportunity to stabilize structures, but also implying a danger that non-specific interactions might occur. Thus, membrane proteins may fold to avoid exposure of strongly hydrogen bonding groups at their lipid exposed surfaces.

Amino Acid Motifs↗
PubMed1999

An O(2n) volume molecular algorithm for Hamiltonian path.

We design volume-efficient molecular algorithms for all problems in #P, using only reasonable biological operations. In particular, we give a polynomial-time 0(2(n)n2log2n)-volume algorithm to compute the number of Hamiltonian paths in an n-node graph. This improves Adleman's celebrated n!-volume algorithm for finding a single Hamiltonian path.

Algorithms↗
PubMed1999

Hyperalgesia due to nerve damage: role of nerve growth factor.

The hypothesis that nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) contribute to hyperalgesia resulting from nerve damage was tested in rats in which the sciatic nerve was partially transected on one side. Administration of antisera raised against NGF and BDNF relieved mechanical and thermal hyperalgesia in these animals. It has been suggested that NGF may elicit hyperalgesia by inducing mast cells to release algesic agents such as serotonin (5-HT). We found that degranulation of mast cells with compound 48/80 relieved mechanical and thermal hyperalgesia produced by nerve damage. We also found that local injection of the 5-HT2A and 5-HT3 receptor antagonists ketanserin and ICS 205-930 into the affected hind paw relieved mechanical hyperalgesia in a dose-dependent fashion. These findings support the idea that in this rat model of hyperalgesia due to peripheral nerve damage, NGF acts on mast cells to induce release of 5-HT, which sensitizes nociceptors. Hyperalgesia due to nerve injury and hyperalgesia due to inflammation may share some common features.

Animals↗
PubMed1996

Structure of DNA-porphyrin complex.

We report the 2.4 A resolution X-ray structure of a complex in which a small molecule flips a base out of a DNA helical stack. The small molecule is a metalloporphyrin, CuTMPyP4 [copper(II) meso-tetra(N-methyl-4-pyridyl)porphyrin], and the DNA is a hexamer duplex, [d(CGATCG)]2. The porphyrin system, with the copper atom near the helical axis, is located within the helical stack. The porphyrin binds by normal intercalation between the C and G of 5' TCG 3' and by extruding the C of 5' CGA 3'. The DNA forms a distorted right-handed helix with only four normal cross-strand Watson-Crick base pairs. Two pyridyl rings are located in each groove of the DNA. The complex appears to be extensively stabilized by electrostatic interactions between positively charged nitrogen atoms of the pyridyl rings and negatively charged phosphate oxygen atoms of the DNA. Favorable electrostatic interactions appear to draw the porphyrin into the duplex interior, offsetting unfavorable steric clashes between the pyridyl rings and the DNA backbone. These pyridyl-backbone clashes extend the DNA along its axis and preclude formation of van der Waals stacking contacts in the interior of the complex. Stacking contacts are the primary contributor to stability of DNA. The unusual lack of van der Waals stacking contacts in the porphyrin complex destabilizes the DNA duplex and decreases the energetic cost of local melting. Thus extrusion of a base appears to be facilitated by pyridyl-DNA steric clashes.

Base Sequence↗
PubMed1996

Clathrate hydrates are poor models of biomolecule hydration.

Clathrate hydrates form the basis of a general model of biomolecule hydration. In clathrate hydrate crystal structures, the size of hydrogen-bonded water rings is highly constrained to five members. The clathrate hydrate model predicts that the size of water rings near biomolecule surfaces is similarly constrained to five members. This report describes a test of this model of biomolecule hydration. We have demonstrated that five-membered water rings are not a general feature of protein or nucleic acid hydration. The clathrate hydrate model appears to be inappropriate for soluble biomolecules.

Base Sequence↗
PubMed1994

Solid-phase derivatization reactions for biomedical liquid chromatography.

Polymeric reagents have been developed for performing off- and on-line derivatizations of numerous organic analytes in HPLC-detection modes. Such reagents utilize ionic or covalent attachment of labile tags that possess specific detector enhancement properties: ultraviolet, electrochemical, fluorescence, and so forth. Specific synthetic procedures have evolved to generate various linkages of the tag to the underlying, polymeric support, usually involving activated ester connections (leashes). The polymer itself may play a number of roles in the nature of the overall reactions, such as hydrophobic-hydrophillic exclusion, pore size restriction, stabilization of the attachment leashes, and protection of the tags from hydrolysis in aqueous media. The basic, underlying chemistry of polymeric reagents has evolved to the point where it is possible to engineer the polymer support itself, the attachment leash, and the various tags that are then transferred to the analyte molecules. These procedures have now reached the stage of commercialization and practical applicability for real-world drugs and bioorganics in complex biofluid type samples. Polymer supported reagents can now be used for direct injection of biofluids with solid-phase (hydrophobic) extraction of the analytes of interest, followed by sample cleanup, derivatization, elution onto the HPLC column, peak compression, gradient HPLC elution, multiple detection, and final data interpretation with quantitation. This review summarizes much or most of what has been described in the scientific literature over the past decade in the various areas where polymeric reagents are being used for derivatization in HPLC and in capillary electrophoresis as well.

Chemistry Techniques, Analytical↗
PubMed1994

Water ring structure at DNA interfaces: hydration and dynamics of DNA-anthracycline complexes.

In crystallographic structures of biological macromolecules, one can observe many hydration rings that originate at one water molecule, pass via hydrogen bonds through several others, and return to the original water molecule. Five-membered water rings have been thought to occur with greater frequency than other ring sizes. We describe a quantitative assessment of relationships between water ring size and frequency of occurrence in the vicinity of nucleic acid interfaces. This report focuses on low-temperature X-ray crystallographic structures of two anthracyclines, adriamycin (ADRI) and daunomycin (DAUN), bound to d(CGATCG) and on several DNA structures published previously by others. We have obtained excellent low-temperature (-160 degrees C, LT) X-ray intensity data for d(CGATCG)-adriamycin and d(CGATCG)-daunomycin with a multiwire area detector. The LTX-ray data sets contain 20% (daunomycin, LT-DAUN) and 35% (adriamycin, LT-ADRI) more reflections than were used to derive the original room-temperature (15 degrees C) structures [Frederick, C.A., Williams, L.D., Ughetto, G., van der Marel, G. A., van Boom, J.H., Rich, A., & Wang, A.H.-J. (1990) Biochemistry 29, 2538-2549]. The results show that five-membered water rings are not preferred over other ring sizes. This assessment is consistent with our observation of broad dispersion W-W-W angles (sigma = 20 degrees). In addition, we report that the thermal mobility, distinct from the static disorder, of the amino sugar of daunomycin and adriamycin is significantly greater than that of the rest of the complex. This mobility implies that if the central AT base pair is switched to a CG base pair, there should be a low energy cost in avoiding the guanine amino group. The energy difference (for the sugar-binding preference) between d(CGTACG) and d(CGCGCG) could be considerably less than 20 kcal/mol, a value proposed previously from computation.

Base Composition↗
PubMed1993

Direct determination of adamantanamine in plasma and urine with automated solid phase derivatization.

A simple, highly sensitive and selective method is described for adamantanamine determination in plasma and urine by high-performance liquid chromatography with fluorescence detection. The method involved a simultaneous extraction and derivatization of biological fluids with a 9-fluoreneacetate (9-FA) solid-phase derivatization reagent. This approach eliminated tedious sample preparation steps and provided automatic derivatization with selective and efficient sample clean-up for direct injection of biological fluids. Derivatized adamantanamine was separated under conventional reversed-phase conditions and determined by fluorescence detection. The optimization and validation of the derivatization method with the 9-FA solid-phase reagent is described.

Acetates↗
PubMed1992

9-Fluoreneacetyl-tagged, solid-phase reagent for derivatization in direct plasma injection.

We describe here a resin-based derivatization reagent, containing a 9-fluoreneacetyl tag on a controlled-pore substrate, for direct injection analysis of amphetamine in plasma. On-line, pre-column derivatization was performed by direction injection of diluted plasma sample into an sodium dodecyl sulfate-containing mobile phase. Amphetamine was trapped in the hydrophobic derivatization column and derivatized at elevated temperature by the activated solid-phase reagent. The derivatized 9-fluoreneacetyl amphetamide was separated by reversed-phase high-performance liquid chromatography with a step gradient and determined by fluorescence detection. The synthesis scheme, characterization, and optimization of the derivatization conditions for the solid-phase reagent are described. The method was evaluated by reproducibility tests and single blind spiking analysis. This solid-phase reagent combined with a surfactant containing mobile phase provided a sensitive and simple procedure for on-line derivatization in direct injection analysis of biological fluids.

Amphetamine↗
PubMed1992

Comparison of 9-fluorenylmethoxycarbonyl and 9-fluoreneacetyl-tagged silica-based derivatization reagents in high-performance liquid chromatography.

Two silica reagents based on a 4-hydroxy-3-nitrobenzoyl backbone were synthesized and characterized with 9-fluorenylmethoxycarbonyl (FMOC) and 9-fluoreneacetyl (FA) tags. These reagents were tested by derivatization of primary and secondary amines. Derivatization conditions such as temperature, time and triethylamine catalyst were tested. The FA-tagged silica reagent showed better performance than the FMOC-tagged silica reagent by a comparison of derivatization efficiencies, stabilities of reagents, and blank reagent interferences with derivatization. Finally, cadaverine and an aliphatic amine mixture were analysed using the FA-tagged reagent by pre-column, off-line derivatization and fluorescence detection.

Acetates↗
PubMed1990

Plasmin-induced platelet aggregation is accompanied by cleavage of aggregin and indirectly mediated by calpain.

We recently reported that thrombin-induced platelet aggregation 1) is accompanied by cleavage of aggregin, a 100-kDa membrane protein and a putative ADP receptor, 2) is indirectly mediated by intracellularly activated calpain, and 3) requires the occupancy of high-affinity thrombin receptors. Because of the similarities between responses after platelet activation induced by thrombin and plasmin (greater than or equal to 1.0 casein unit/ml), we investigated whether or not plasmin-induced platelet aggregation proceeds by the same mechanism that underlies thrombin-induced platelet aggregation. We found that the rate of plasmin-induced aggregation of washed intact platelets and that of platelets modified by 5'-p-fluorosulfonylbenzoyladenosine (FSBA, an affinity analogue of ADP, which covalently modifies aggregin) were similar, indicating that the aggregation is independent of the ADP effect. Plasmin completely cleaved [3H]FSBA-labeled aggregin in intact platelets. A mixture of metabolic inhibitors (2-deoxy-D-glucose, gluconolactone, and antimycin A) completely inhibited plasmin-induced platelet aggregation and plasmin-induced cleavage of aggregin, demonstrating that an energy-requiring step is involved in the reaction. The synthetic hexapeptide affinity reagent Phe-Gln-Val-Val-Cys(NpyS)-Gly-NH2 (NpyS = 3-nitro-2-thiopyridine), a potent and specific inhibitor of thrombin-induced platelet aggregation and platelet calpain, completely inhibited plasmin-induced platelet aggregation and plasmin-induced cleavage of aggregin. These results suggest that, like thrombin, plasmin-induced platelet aggregation is accompanied by the cleavage of aggregin and these responses are indirectly mediated by the intracellularly activated calpain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Ultrasonographic diagnosis of achalasia.

A perspective ultrasonographic study on 10 cases of achalasia showed characteristic ultrasonographic features: dilation and persistent water retention of the gastroesophageal vestibule, symmetrical parietal thickening, and delayed or intermittent opening of the cardiac orifice after drinking. We suggest that ultrasonography should play an important role in clinical management of achalasia. If the ultrasonographic features of achalasia were known, the misdiagnosis of achalasia for cardiac carcinoma could be avoided. When an infiltrating cardiac carcinoma found to be smoothly narrowing and difficult to distinguish from achalasia radiologically, an ultrasonogram may be helpful to make a correct diagnosis.

Adenocarcinoma↗
PubMed1989

Cleavage of a 100 kDa membrane protein (aggregin) during thrombin-induced platelet aggregation is mediated by the high affinity thrombin receptors.

Thrombin-induced platelet aggregation is accompanied by cleavage of aggregin, a surface membrane protein (Mr = 100 kDa), and is mediated by the intracellular activation of calpain. We now find that agents that increase intracellular levels of platelet cAMP by stimulating adenylate cyclase, also inhibit thrombin binding and platelet activation by destabilizing thrombin receptors on the platelet surface. Iloprost (a stable analog of PGI2) and forskolin each completely inhibited platelet aggregation by 2 nM thrombin and markedly decreased cleavage of aggregin. Thrombin inactivated by D-phenylalanine-L-prolyl-L-arginine chloromethyl ketone (PPACK-thrombin) binds to the highest affinity site for thrombin on the platelet surface, but thrombin modified by N alpha-tosyl-L-lysine chloromethylketone (TLCK-thrombin) does not. We now demonstrate that preincubation of platelets with PPACK-thrombin blocked platelet aggregation and cleavage of aggregin induced by 2 nM thrombin. In contrast, TLCK-thrombin neither blocked platelet aggregation nor the cleavage of aggregin. These results show that a) platelet aggregation and cleavage of aggregin by thrombin (2nm) involves the occupancy of high affinity alpha-thrombin receptors on the platelet surface, and b) stimulators of adenylate cyclase which increase cAMP, inhibit thrombin-induced platelet aggregation and cleavage of aggregin by mechanisms which include inhibiting the binding of thrombin to its receptors.

Calpain↗
PubMed1989

Thrombin-induced platelet aggregation involves an indirect proteolytic cleavage of aggregin by calpain.

5'-p-Fluorosulfonylbenzoyl adenosine (FSBA), a nucleotide analog of ADP, has been shown to inhibit ADP-induced shape change, aggregation and exposure of fibrinogen binding sites concomitant with covalent modification of a single surface membrane polypeptide of Mr 100,000 (aggregin). Since thrombin can aggregate platelets which have been modified by FSBA and are refractory to ADP, we tested the hypothesis that thrombin-induced platelet aggregation might involve cleavage of aggregin. At a low concentration of thrombin (0.05 U/ml), platelet aggregation, exposure of fibrinogen receptors and cleavage of aggregin in FSBA-modified platelets did not occur, indicating ADP dependence. In contrast, incubation of [3H]FSBA-labeled intact platelets with a higher concentration of thrombin (0.2 U/ml) resulted in cleavage of radiolabeled aggregin, aggregation, and exposure of fibrinogen binding sites. Under identical conditions, aggregin in membranes isolated from [3H]FSBA-labeled platelets was not cleaved by thrombin. Thrombin-induced platelet aggregation and cleavage of aggregin were concomitantly inhibited by a mixture of 2-deoxy-D-glucose, D-gluconic acid 1,5-lactone, and antimycin A. These results suggest that thrombin cleaves aggregin indirectly by activating an endogeneous protease. Thrombin is known to elevate intracellular Ca2+ concentration and thereby activates intracellular calcium dependent thiol proteases (calpains). In contrast to serine protease inhibitors, calpain inhibitors including leupeptin, antipain, and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (chelator of Ca2+) inhibited platelet aggregation and cleavage of aggregin in [3H]FSBA-labeled platelets. Leupeptin, at a concentration of 10-20 microM, used in these experiments, did not inhibit the amidolytic activity of thrombin, thrombin-induced platelet shape change, or the rise in intracellular Ca2+. Purified platelet calpain II caused aggregation of unmodified and FSBA-modified platelets and cleaved aggregin in [3H]FSBA-labeled platelets as well as in isolated membranes. The latter is in marked contrast to the action of thrombin on [3H]FSBA-labeled membranes. Thus, thrombin-induced platelet aggregation may involve intracellular activation of calpain which proteolytically cleaves aggregin thus unmasking latent fibrinogen receptors, a necessary prerequisite for platelet aggregation.

Adenosine↗