Biomedical subjects
Publications and source records attributed to K He.
4(S)-Chloro-4-deoxygigantetrocin A and 4(S), 18-dichloro-4,18-dideoxyasimilobin were obtained by treating gigantetrocin A with triphenylphosphine and CCl4. The structures were determined by spectroscopic methods. The chlorinated compounds show decreased bioactivities in the brine shrimp lethality test and against human tumor cell lines.
Three new adjacent bis-tetrahydrofuran ring Annonaceous acetogenins with four hydroxy groups, bullatetrocin (1), 10-hydroxyasimicin (2), and 10-hydroxytrilobacin (3), were isolated by activity-directed fractionation from the stem bark of Asimina triloba. Their structures were established on the basis of chemical and spectral evidence. The absolute stereochemistry at the C-10 hydroxy position was determined by converting 2 and 3 to their ketolactone isomers, 2,4-cis/trans 10-hydroxyasimicinones and 2,4-cis/trans 10-hydroxytrilobacinones, respectively. The bioactivities of the new compounds against brine shrimp larvae and six human solid-tumor cell lines are reported, and structure-activity relationships between trihydroxylated and tetrahydroxylated acetogenins are discussed. In addition to 1-3, gigantetrocin A, 2,4-cis/trans-gigantetrocin A-ones, annonacin, and annonacin A were also isolated for the first time from this species.
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Magainin 2 is a 23-residue antibiotic peptide found in the skin of Xeonpus laevis (African clawed frog). It belongs to a broad class of alpha-helical peptides which interact directly with the lipid bilayer. Very little is presently known about the nature of this peptide/lipid interaction on the molecular level. We have performed a sequence of lamellar X-ray diffraction experiments to provide some insight into the nature of this interaction. We have found that, at concentrations below the critical concentration for lysis, the peptide causes the membrane thickness to decrease roughly in proportion to the peptide concentration. We further show that this thinning is consistent with a model where the peptide adsorbs within the headgroup region of the lipid bilayer at these concentrations. The energy cost of this thinning may also explain why the peptide inserts at high concentrations. We have already shown that a similar interaction exists for alamethicin interacting with diphytanoylphosphatidylcholine, and it should hold for a wide variety of peptide/lipid systems.
Antimicrobial peptides isolated from the host defense systems of animals have been shown to exert their activity directly on the lipid bilayer of cell membranes, but the antimicrobial mechanisms are not clear, due chiefly to the difficulty of discerning the high-order structures formed by these peptides in membranes. Previously we have shown that these peptides insert into the membrane when their concentrations exceed a lipid-dependent critical value. With neutron in-plane scattering we now show that inserted alamethicin creates aqueous pores approximately greater than 18 A in diameter. The density of pores is consistent with the assumption that all of the alamethicin is involved in pore formation. Pores were not detected below the critical concentration. Thus concentration-dependent pore formation appears to be the molecular mechanism of antimicrobial action.
Although microglia are the only cells found to be productively infected in the central nervous system of acquired immunodeficiency disease syndrome (AIDS) patients, there is extensive white and gray matter disease nonetheless. This neuropathogenesis is believed to be due to indirect mechanisms other than infection with human immunodeficiency virus 1 (HIV-1). Cytokines and toxic small molecules have been implicated in the clinical and histopathological findings in CNS AIDS. Previously, we have demonstrated in rodent glial cultures the presence of biologically active epitopes of gp120 and gp41 that are capable of inducing interleukin 1 and tumor necrosis factor alpha. In this study, we map the HIV-1 envelope epitopes that induce nitric oxide, inducible nitric oxide synthase, interleukin 1, and tumor necrosis factor alpha in human glial cultures. Epitopes in the carboxy terminus of gp120 and the amino terminus of gp41 induce these proinflammatory entities. In addition, we compare HIV-1 infection and pathology in glial cells derived from human brain taken at different states of maturation (fetal, neonatal, and adult brain) in an effort to address some of the clinical and histological differences seen in vivo. This study demonstrates that, in the absence of virus infection and even in the absence of distinct viral tropism, human glia respond like rodent glia to non-CD4-binding epitopes of gp120/gp41 with cytokine and nitric oxide production. Differences among fetal, neonatal, and adult glial cells' infectivity and cytokine production indicate that, in addition to functional differences of glia at different stages of development, cofactors in vitro and in vivo may also be critical in facilitating the biological responses of these cells to HIV-1.
The pathology in central nervous system (CNS) AIDS suggests that direct infection with HIV-1 is not required for changes in glia and neurons. Induction of a variety of pathological responses in vitro in rodent brain cultures also suggests that CD4 is not the receptor for HIV-1 in the brain, given that human and rodent CD4 are not homologous. This implies that the epitopes on HIV-1 which bind glia and activate them are novel, non-CD4-binding domains. We have therefore mapped the envelope (env) regions required for production in rat glial cultures of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) which we hypothesize are important in CNS AIDS. Serially truncated deletion mutants from the gp120/gp41 carboxy terminus representing folded, glycosylated recombinant env proteins were expressed in HeLa cells via a vaccinia virus vector. These proteins, linear gp120/gp41 peptides, as well as polyclonal and monoclonal antibodies reactive to defined regions of gp120/gp41 were used to map the epitopes involved in production of IL-1 and TNF alpha. Compared to HeLa cell and wild-type vaccinia virus controls, the vaccinia recombinant env protein gp160 containing cleaved gp120 and gp41 induced both IL-1 and TNF alpha. If gp160 was not cleaved into gp120 and gp41, IL-1 but not TNF alpha induction was reduced. Peak production of TNF alpha by gp120/gp41 was at 4 h while IL-1 production was still significantly elevated at 44 h at the highest concentrations of env protein. Using the truncation deletions, the V3 loop of gp120 appeared to be critical for IL-1. Glycosylation and folding of V3 is probably important in IL-1 induction since a V3 peptide was not as active. While removal of glycosylated, folded V4 and C4 regions had no effect on IL-1, linear peptides in the region from the V4 loop to the C4 domain were strong inducers of IL-1. Non-glycosylated, linear V4 loop peptide induced more IL-1 than the V4 in protein generated in HeLa cells, suggesting that glycosylation and/or conformational structures sequester V4 inducer epitopes. Using the truncation deletions, the carboxy terminus region (V4-C5) of gp120 as well as gp41 were shown to be critical for TNF alpha production. Peptides representing linear epitopes in the V3 loop, C5 domain of gp120, and the ectodomain of gp41 were all strong inducers of TNF alpha; a protein representing almost the entire gp41 was the strongest inducer of TNF alpha.(ABSTRACT TRUNCATED AT 400 WORDS)
A variety of amphiphilic helical peptides have been shown to exhibit a transition from adsorbing parallel to a membrane surface at low concentrations to inserting perpendicularly into the membrane at high concentrations. Furthermore, this transition has been correlated to the peptides' cytolytic activities. X-ray lamellar diffraction of diphytanoyl phosphatidylcholine-alamethicin mixtures revealed the changes of the bilayer structure with alamethicin concentration. In particular, the bilayer thickness decreases with increasing peptide concentration in proportion to the peptide-lipid molar ratio from as low as 1:150 to 1:47; the latter is near the threshold of the critical concentration for insertion. From the decreases of the bilayer thickness, one can calculate the cross sectional expansions of the lipid chains. For all of the peptide concentrations studied, the area expansion of the chain region for each adsorbed peptide is a constant 280 +/- 20 A2, which is approximately the cross sectional area of an adsorbed alamethicin. This implies that the peptide is adsorbed at the interface of the hydrocarbon region, separating the lipid headgroups laterally. Interestingly, the chain disorder caused by a peptide adsorption tends to spread over a large area, as much as 100 A in diameter. The theoretical basis of the long range nature of bilayer deformation is discussed.
Using oriented circular dichroism, we have found that magainin adopts an alpha-helical conformation with two distinct orientations when interacting with a lipid bilayer. At low concentrations, magainin is absorbed parallel to the membrane surface. However, at high concentrations, magainin is inserted into the membrane. This transition occurs at roughly the same critical concentration required for cytolytic activity, implying that the membrane insertion is responsible for magainin's cell-lysing activity.
An analogue of gramicidin A (gA) was synthesized with the formyl group replaced by a BOC group. The analogue (BOC-gA) exhibited single channel conduction, but the channel is 5-order-of-magnitude destabilized relative to the gA channel. Hydrated mixtures of gramicidin and dilauroyl phosphatidylcholine in the molar ratio of 1:10 were prepared into uniformly aligned multiple bilayers, and X-ray scattering with the momentum transfer in the plane of the membrane was measured. Analysis with the help of computer simulations showed that 70% of BOC-gA are monomers. Thus for the first time it was shown that gramicidin monomers are stable inside the monolayers of a lipid membrane. Furthermore, the monomers have the same beta helical conformation as the dimeric channel. The result suggests the possibility that when a gramicidin channel is closed, it dissociates into two monomers floating in opposite monolayers.
A natural substance, telluroamino acid, was discovered in tellurium yeast by GC and GC/MS. This substance was obtained for the first time by means of the assimilation of inorganic tellurium by yeast, having 600 ppm of total Te and 150 ppm of telluroamino acid. Some rules for such an assimilation were found: the content of total Te and telluroamino acid in tellurium-yeast is closely related to the kind of culture media used and is proportional to the concentration of Te in culture media. This discovery is of theoretical and practical significance, especially for the production by a simpler microbiological synthesis of rare and expensive organic tellurium compounds which hopefully will prove to be anticarcinogens like the selenium-yeast, and for explaining the biochemical effect of tellurium on the human body.
We demonstrate a technique for measuring x-ray (or neutron) scattering with the momentum transfer confined in the plane of membrane, for the purpose of studying lateral organization of proteins and peptides in membrane. Unlike freeze-fracture electron microscopy or atomic force microscopy which requires the membrane to be frozen or fixed, in-plane x-ray scattering can be performed with the membrane maintained in the liquid crystalline state. As an example, the controversial question of whether gramicidin forms aggregates in membrane was investigated. We used dilauroylphosphatidylcholine (DLPC) bilayers containing gramicidin in the molar ratio of 10:1. Very clear scattering curves reflecting gramicidin channel-channel correlation were obtained, even for the sample containing no heavy atoms. Thallium ions bound to gramicidin channels merely increase the magnitude of the scattering curve. Analysis of the data shows that the channels were randomly distributed in the membrane, similar to a computer simulation of freely moving disks in a plane. We suggest that oriented proteins may provide substantial x-ray contrast against the lipid background without requiring heavy-atom labeling. This should open up many possible new experiments.
By using Western blot method, we studied the antihistone fraction antibodies in sera of patients with rheumatic diseases. After analysed with SDS-PAGE, the histone could be separated into H1, H3, H2B, H2A and H4 fractions. The positive rate of anti-histone fraction antibodies was highest (82.1%) in systemic lupus erythematosus (SLE) patients, especially in active stage. In 28 SLE patients, the positive rates of anti-H2B, -H1, -H3, -H2A and -H4 were 78.6%, 60.7%, 50.0%, 35.7% and 7.1% respectively. The results revealed that the determination of anti-histone fraction antibodies was beneficial to the diagnosis of SLE.
Seven human liver samples were collected in this study. Five of them were obtained from donors died from non-disease accident, one was taken from part of the normal tissue of a liver specimen obtained by partial hepatectomy in a case of hepatic cavernous hemangioma and the last one from postmortem died by road accident. All the liver pieces were stored at -75 degrees C immediately after removed from the body. Liver microsomes was prepared by differential ultracentrifugation. The contents of cytochrome P450 and cytochrome b5 in Chinese adult liver microsomes were 0.36 +/- 0.08 and 0.23 +/- 0.05 nmol.mg-1 protein (n = 7). The activities of aminopyrine and ethylmorphine N-demethylase, 7-ethoxycoumarin-O-deethylase, nifedipine oxidase and (-)-praziquantel hydroxylase were 1.07 +/- 0.23, 1.82 +/- 0.31, 0.30 +/- 0.10, 0.43 +/- 0.18 and 0.69 +/- 0.43 nmol.mg-1.min-1 (n = 7), respectively.
The stereoselective binding of praziquantel (PQT) enantiomers to plasma proteins was studied by equilibrium dialysis. The binding of (+/-)-PQT and its enantiomers to bovine serum albumin (BSA) and rabbit plasma was unsaturable when initial drug concentrations from 1 to 32 mumol/L were used. The binding capacity (nK) of (+/-)-PQT to BSA (1.47 or 5.88 x 10(-4) mol/L) was greater than that of (-)-PQT with a (+)-/(-)-PQT ratio of 1.25 or 1.26, respectively. However, the preferential binding of (-)-PQT to rabbit plasma was found to be 73.7 +/- 4.4% for (-)-isomer and 58.3 +/- 10.1% for (+)-isomer (n = 8, P less than 0.05). The binding of (+)- and (-)-PQT to human plasma was 81.9 +/- 4.2% and 83.2 +/- 6.9% (n = 10, P greater than 0.05) respectively, which seems to be not stereoselective. These results indicate that there are species differences in the stereoselective binding of PQT enantiomers to plasma proteins.
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The effect of endotoxin on human platelet was studied in vitro. E Coli O55B5 endotoxin inhibited spontaneous platelet aggregation but had no effect on platelet aggregation by epinephrine and the release of contents from dense granule, alpha-granule of platelet. The endotoxin also elevated cAMP and increased the activity of calmodulin in platelet, which might account for the effect of the endotoxin on platelet. In addition, a reduced concentration of 5-HT in the supernate of endotoxin group was also observed.