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Biomedical subjects

L P Liu

Publications and source records attributed to L P Liu.

At least 19 recordsLinked to original sources
PubMed1999

The portosystemic shunt protects liver against ischemic reperfusion injury.

BACKGROUND: The goal of this study was to characterize the importance of splanchnic viscera in liver ischemic reperfusion injury and to enhance the tolerance of liver to warm ischemia injury with portosystemic shunt. METHODS: The hepatic blood flow of male Sprague Dawley rats was subjected to 45, 60, 120, and 150 min liver warm ischemia with or without portosystemic shunt (splenic-caval shunt). The production of tumor necrosis factor a (TNFa), nuclear factor-kappaB activation, inducible NO synthase (iNOS) expression, and apoptosis were examined. RESULTS: A total of 67% of rats with 45 min liver warm ischemia (n=6) and 100% of rats with 60 min liver warm ischemia (n=6) died within 1 day. However, all rats with 120 min (n=8) liver warm ischemia in splenic-caval shunt group survived for over 1 day, 6/8 for over 3 days, and 5/8 for over 5 days without significant histological changes of the liver. Serum tumor necrosis factor levels in liver warm ischemic rats were increased, This increase was significantly reversed after portosystemic shunt. After challenge with lipopolysaccharide (1 mg/kg, p.v.), naive rats survived for over 5 days (n=4) with the peak value of rat tumor necrosis factor (240 pg/ml) at 90 min. In contrast, all rats died within one day (n=5) with the peak value of rat tumor necrosis factor a (465 pg/ml) at 45 min after administration of lipopolysaccharide in the rats with liver warm ischemia plus splenic-caval shunt. iNOS expression and nuclear factor-kappaB activation were very strongly increased in the hepatocytes after liver warm ischemia with portosystemic shunt, compared with liver ischemia without portosytemic shunt. CONCLUSIONS: We conclude that the splanchnic viscera can contribute to liver ischemic reperfusion injury. Portosystemic shunt enhances the tolerance of liver to warm ischemia through the protective role of iNOS and nuclear factor-kappaB (NF-kappaB).

Animals↗
PubMed1999

Combining hydrophobicity and helicity: a novel approach to membrane protein structure prediction.

In spite of the overwhelming numbers and critical biological functions of membrane proteins, only a few have been characterized by high-resolution structural techniques. From the structures that are known, it is seen that their transmembrane (TM) segments tend to fold most often into alpha-helices. To evaluate systematically the features of these TM segments, we have taken two approaches: (1) using the experimentally-measured residence behavior of specifically designed hydrophobic peptides in RP-HPLC, a scale was derived based directly on the properties of individual amino acids incorporated into membrane-interactive helices: and (2) the relative alpha-helical propensity of each of the 20 amino acids was measured in the organic non-polar environment of n-butanol. By combining the resulting hydrophobicity and helical propensity data, in conjunction with consideration of the 'threshold hydrophobicity' required for spontaneous membrane integration of protein segments, an approach was developed for prediction of TM segments wherein each must fulfill the dual requirements of hydrophobicity and helicity. Evaluated against the available high-resolution structural data on membrane proteins, the present combining method is shown to provide accurate predictions for the locations of TM helices. In contrast, no segment in soluble proteins was predicted as a 'TM helix'.

1-Butanol↗
PubMed1999

Perspective: peptides as mimics of transmembrane segments in proteins.

Peptide-based approaches to protein structure within membranes have proven enormously valuable. When one focusses on the detailed manner through which membrane proteins actually traverse the cell bilayer, a simple observation emerges: helical peptide segments of 20 amino acids each constitute the only tangible connection between the inside and outside of the cell. Thus, a major step towards understanding the key relationships between biological function and membrane protein structure can be taken through characterization, by composition, sequence, chain length, hydrophobicity and conformation, of hydrophobic peptides designed as mimics of transmembrane segments.

Amino Acid Sequence↗
PubMed1998

Uncoupling hydrophobicity and helicity in transmembrane segments. Alpha-helical propensities of the amino acids in non-polar environments.

Although the chains of amino acids in proteins that span the membrane are demonstrably helical and hydrophobic, little attention has been paid toward addressing the range of helical propensities of individual amino acids in the non-polar environment of membranes. Because it is inappropriate to apply soluble protein-based structure prediction algorithms to membrane proteins, we have used de novo designed peptides (KKAAAXAAAAAXAAWAAXAAAKKKK-amide, where X indicates one of the 20 commonly occurring amino acids) that mimic a protein membrane-spanning domain to determine the alpha-helical proclivity of each residue in the isotropic non-polar environment of n-butanol. Peptide helicities measured by circular dichroism spectroscopy were found to range from theta222 = -17,000 degrees (Pro) to -38,800 degrees (Ile) in n-butanol. The relative helicity of each amino acid is shown to be well correlated with its occurrence frequency in natural transmembrane segments, indicating that the helical propensity of individual residues in concert with their hydrophobicity may be a key determinant of the conformations of protein segments in membranes.

Amino Acid Sequence↗
PubMed1998

Cystic fibrosis transmembrane conductance regulator: expression and helicity of a double membrane-spanning segment.

The gene responsible for cystic fibrosis encodes a membrane protein--the 1480-residue cystic fibrosis transmembrane conductance regulator (CFTR)--in which membrane-based CF-phenotypic mutants alter pore structure and/or impair ion transport. We report the preparation in milligram quantities and conformational characterization of a polypeptide comprised of CFTR transmembrane (TM) segments 3-4, a putative 'helical hairpin' portion of the CFTR TM1-6 domain. The TM segment 3-4 of CFTR was expressed in E. coli as a fusion protein linked to the C-terminus of His-tagged thioredoxin. Nickel chelate affinity chromatography, followed by release from the carrier by digestion with thrombin protease, gave free CFTR(TM3-4). Monitoring of the folding properties and conformational state(s) of the TM3-4 polypeptide using circular dichroism spectroscopy indicated a partial alpha-helical conformation in aqueous buffer, with up to 30% increase in alpha-helical content observed in membrane-mimetic environments.

Cell Membrane↗
PubMed1998

Guidelines for membrane protein engineering derived from de novo designed model peptides.

Notwithstanding great advances in the engineering and structural analysis of globular proteins, relatively limited success has been achieved with membrane proteins--due largely to their intrinsic high insolubility and the concomitant difficulty in obtaining crystals. Progress with de novo synthesis of model membrane-interactive peptides presents an opportunity to construct simpler peptides with definable structures, and permits one to approach an understanding of the properties of the membrane proteins themselves. In the present article, we review how our laboratory and others have used peptide approaches to assess the detailed interactions of peptides with membranes, and primary folding at membrane surfaces and in membranes. Structural studies of model peptides identified the existence of a "threshold hydrophobicity," which controls spontaneous peptide insertion into membranes. Related studies of the relative helicity of peptides in organic media such as n-butanol indicate that the helical propensity of individual residues--not simply their hydrophobicity--may dictate the conformations of peptides in membranes. The overall experimental results provide fundamental guidelines for membrane protein engineering.

Amino Acid Sequence↗
PubMed1998

Partial purification of embryotrophic factors from human oviductal cells.

Embryotrophic factors from human oviductal cells were partially purified by liquid chromatographic methods. The conditioned medium from human oviductal cell culture was fractionated successively by concanavalin A (Con-A) affinity chromatography, ion-exchange chromatography and gel filtration. The presence of the embryotrophic activity in the eluates was determined by the stimulatory effects on the development of mouse embryos in vitro. The fraction that did not bind to the lectin Con-A possessed no embryotrophic activity. Ion-exchange chromatography separated the glycoproteins that bound to Con-A into five fractions. Three of them significantly enhanced blastulation as well as conceptus formation. Gel filtration further separated these embryotrophic fractions into five fractions. Three of them with molecular weights of 154 +/- 1, 164 +/- 0.2 and 207 +/- 0.3 kDa significantly stimulated blastulation of mouse embryos. The results of this study demonstrated that several embryotrophic factors with different biochemical properties contributed to the embryotrophic effect of the human oviductal cell/mouse embryo co-culture system.

Animals↗
PubMed1998

Differential efficacy of passive immunisation against infection by Lyme disease spirochaetes transmitted by partially fed vector ticks.

The efficacy of passive immunisation against tick-transmitted Lyme disease spirochaetal infection was determined in relation to the duration of previous feeding of infected vector ticks. Thus, mice challenged with spirochaete-infected unfed or partially fed nymphal ticks were passively immunised with monoclonal and polyclonal antibodies against the Lyme disease spirochaete (Borrelia burgdorferi) at various intervals after tick attachment. Spirochaetal infection in challenged mice and engorged ticks was verified by xenodiagnosis and indirect immunofluorescent antibody assay, respectively. Although tick-transmitted spirochaetal infection could be aborted by anti-OspA antibodies and hyperimmune antiserum, nearly all immunised mice challenged with infected ticks that had previous 36-h attachment became infected. More than 72% of the nymphal ticks used in this challenge retained their B. burgdorferi infection after engorgement on mice immunised with anti-spirochaete antibodies, and their subsequent infectivity to mice remained effective. It is concluded that a higher efficiency of transmission by partially fed infected nymphs and a lower efficacy of passive immunisation against infection result from an effect of previous feeding of infected ticks that activates antigenic change and enables the spirochaetes to circumvent OspA-based humoral immunity.

Animals↗
PubMed1998

Effect of procainamide on ultrastructure of blood platelet in rabbits.

AIM: To study the effect of procainamide (PA) on the ultrastructure of blood platelets. METHODS: Arachidonic acid was added to PA-treated platelet-rich plasma to induce platelet aggregation. The 50-nm sections were examined with a transmission electron microscope. RESULTS: PA 8.5-136 mumol.L-1 markedly inhibited changes of pseudopods, alpha-granules, dense granules, glycogens, open canalicular system, and dense tubular system. CONCLUSION: PA markedly inhibited the changes of ultrastructure of blood platelet and releasing response.

Animals↗
PubMed1997

Anionic phospholipids modulate peptide insertion into membranes.

While the insertion of a hydrophobic peptide or membrane protein segment into the bilayer can be spontaneous and driven mainly by the hydrophobic effect, anionic lipids, which comprise ca. 20% of biological membranes, provide a source of electrostatic attractions for binding of proteins/peptides into membranes. To unravel the interplay of hydrophobicity and electrostatics in the binding of peptides into membranes, we designed peptides de novo which possess the typical sequence Lys-Lys-Ala-Ala-Ala-X-Ala-Ala-Ala-Ala-Ala-X-Ala-Ala-Trp-Ala-Ala-X-Ala-Al a-Ala-Lys-Lys-Lys-Lys-amide, where X residues correspond to "guest" residues which encompass a range of hydrophobicity (Leu, Ile, Gly, and Ser). Circular dichroism spectra demonstrated that peptides were partially (40-90%) random in aqueous buffer but were promoted to form 100% alpha-helical structures by anionic lipid micelles. In neutral lipid micelles, only the relatively hydrophobic peptides (X = L and I) spontaneously adopted the alpha-helical conformation, but when 25% of negatively charged lipids were mixed in to mimic the content of anionic lipids in biomembranes, the less hydrophobic (X = S and G) peptides then formed alpha-helical conformations. Consistent with these findings, fluorescence quenching by the aqueous-phase quencher iodide indicated that in anionic (dimyristoylphosphatidylglycerol) vesicles, the peptide Trp residue was buried in the lipid vesicle hydrophobic core, while in neutral (dimyristoylphosphatidylcholine) vesicles, only hydrophobic (X = L and I) peptides were shielded from the aqueous solution. Trp emission spectra of peptides in the presence of phospholipids doxyl-labeled at the 5-, 7-, 10-, 12-, and 16-fatty acid positions implied not only a transbilayer orientation for inserted peptides but also that mixed peptide populations (transbilayer + surface-associated) may arise. Overall results suggest that for hydrophobic peptides with segmental threshold hydrophobicity below that which promotes spontaneous membrane insertion, primary electrostatic attractions provided by anionic phospholipids become essential for peptide binding and insertion to membranes.

Amino Acid Sequence↗
PubMed1997

Human babesiosis in Taiwan: asymptomatic infection with a Babesia microti-like organism in a Taiwanese woman.

An asymptomatic Babesia infection was confirmed by laboratory diagnoses. The intraerythrocytic protozoan (designed TW1) isolated from a 51-year-old Taiwanese woman appeared to be morphologically consistent with small-form piroplasm, and measurements indicated that it had a body size of 1.5 to 2.5 microm in diameter. The typical features of ring, binary, and tetrad forms were observed in Giemsa-stained thin blood smears. A persistent and low-grade parasitemia was established after hamster inoculation. Indirect immunofluorescent-antibody reactivities indicate that this strain (TW1) of Babesia was serologically related to, but not identical to, the Babesia species (B. microti) that infects rodents. Antibody titers in the patient's sera combined with the clinical symptoms suggested that the present case was a chronic and subclinical babesial infection. A neighborhood human serologic survey indicated that the infection may have been acquired accidentally from an infected rodent and localized within the same family. Indeed, rodents from areas around the neighborhood were trapped, and a high prevalence (83%) of babesial infection was observed. The possible vector responsible for the transmission remains to be identified.

Animals↗
PubMed1996

Accelerated infectivity of tick-transmitted Lyme disease spirochetes to vector ticks.

We determined whether the span of infectivity of Lyme disease spirochetes (Borrelia burgdorferi) to vector ticks varies with the mode of infection in laboratory mice. Noninfected larval deer ticks were permitted to feed on two strains of spirochete-infected mice that had been naturally (via tick bite) and parenterally (via needle injection) infected with B. burgdorferi 2, 4, or 8 weeks earlier, and engorged ticks were dissected and examined for spirochetes by direct immunofluorescence microscopy. After initial infection, spirochetal infectivity to ticks was less efficient in needle-infected mice than in mice infected via tick bites. Tick-transmitted spirochetes develop more rapidly from the skin of infected mice and do not induce a strong antispirochete antibody response during the early stage of infection.

Animals↗
PubMed1996

Angiotensin II stimulates T-type Ca2+ channel currents via activation of a G protein, Gi.

Angiotensin II (ANG II) is the most potent and the most physiologically important stimulator of aldosterone synthesis and secretion from the adrenal zona glomerulosa. Because steroidogenesis by adrenal glomerulosa (AG) cells is mediated in part by Ca2+ influx through T- and L-type Ca2+ channels, we evaluated whether T-type Ca2+ channels are regulated by ANG II. We observe that ANG II enhances T-type Ca2+ current by shifting the voltage dependence of channel activation to more negative potentials. This shift is transduced by the ANG II type 1 receptor. The effect of the hormone is not mediated by Ca2+/calmodulin-dependent protein kinase II (CaMKII) as it is not prevented by CaMKII(281-302), a peptide inhibitor of the catalytic region of the kinase. Rather, this shift is mediated by the activation of a G protein, Gi, because it is abolished by cell pretreatment with pertussis toxin and by cell dialysis with a monoclonal antibody generated against recombinant Gi alpha. This effect of ANG II on T-type Ca2+ channels should increase Ca2+ entry in AG cells at physiologically relevant voltages and result in a sustained increase in aldosterone secretion.

Angiotensin II↗
PubMed1995

Human oviductal cells produce high molecular weight factor(s) that improves the development of mouse embryo.

The coculture effects of human oviductal cells on mouse embryo development in vitro were studied. Pronuclear stage mouse zygotes were cocultured with different cell types, or then cultured either in medium alone (control), spent medium from oviductal cell culture (conditioned medium) or high molecular weight fractions (> 10 and > 100 kDa) of the conditioned medium (reconstituted medium). Embryotrophic activities were compared between these groups in terms of percentage of morula and blastocyst formation, and cell count at the blastocyst stage. The mouse embryos developed better in oviductal cell coculture than in fibroblast coculture and medium alone culture. Conditioned medium and its reconstituted medium also provided a significant enhancement of embryo development in vitro when compared with the control medium culture, suggesting the production of high molecular weight embryotrophic factor(s) by the oviductal cells. The high molecular weight embryotrophic activity accumulated with the duration of conditioning could be serially diluted, and was abolished by heat or trypsin treatment. Replacing bovine serum albumin with polyvinyl alcohol in the culture medium did not affect the production of this high molecular weight embryotrophic activity by oviductal cells.

Animals↗
PubMed1995

Differential spirochetal infectivities to vector ticks of mice chronically infected by the agent of Lyme disease.

We determined whether the infectivity of the Lyme disease spirochete (Borrelia burgdorferi) to vector ticks varies with the duration of infection in laboratory mice. Thus, noninfected nymphal deer ticks were permitted to feed on two strains of early (2 months after infection) and late (8 months after infection) spirochete-infected mice. The attached ticks were removed from their hosts at specified time intervals and were thereafter examined for spirochetes by direct immunofluorescence microscopy. Spirochetes can be acquired by nymphal ticks as fast as 8 h after attachment. More than 80% of the attached ticks acquired spirochetal infection within 48 h after feeding on early spirochete-infected mice. In contrast, spirochetal infectivity to ticks was less than 50% after feeding on late spirochete-infected mice. The overall infectivity of spirochete-infected mice to ticks correlated with the duration of tick attachment. In addition, there was no adverse effect on the spirochetal infectivity to ticks by high levels of host antibody against spirochetes, and no obvious differences in infectivity to ticks was observed by the site of tick feeding. We conclude that the span of spirochetal infectivity to ticks varies with the duration of infection in mice and suggest that spirochetes may persist and may be evenly distributed in the skin of infected hosts, regardless of prominent host immunity.

Animals↗
PubMed1995

Ca2+/calmodulin-dependent protein kinase II activation and regulation of adrenal glomerulosa Ca2+ signaling.

We recently reported that elevations in the intracellular Ca2+ concentration ([Ca2+]i) enhance low-voltage-activated, T-type, Ca2+ channel activity via Ca2+/calmodulin-dependent protein kinase II (CaMKII). Here, we document CaMKII activity in bovine adrenal glomerulosa (AG) cells and assess the importance of CaMKII in depolarization-induced Ca2+ signaling. AG cell extracts exhibited kinase activity toward a CaMKII-selective peptide substrate that was dependent on both Ca2+ [half-maximal concentration for Ca2+ activation (K0.5) = 1.5 microM] and calmodulin (K0.5 = 46 nM) and was sensitive to a calmodulin antagonist and a CaMKII peptide inhibitor. On cell treatment with elevated extracellular potassium (10-60 mM) or angiotensin II, Ca(2+)-independent CaMKII activity increased to 133-205% of basal activity. Ca(2+)-independent kinase activity in agonist-stimulated extracts was inhibited by the CaMKII inhibitor peptide, 1(-)[N,O-bis(1,5- isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a cell-permeable inhibitor of CaMKII, reduced the agonist-induced stimulation of Ca(2+)-independent CaMKII activity. KN-62 also diminished depolarization-induced increases in [Ca2+]i without affecting the membrane potential. These observations suggest that CaMKII is activated in situ by aldosterone secretagogues and augments Ca2+ signaling through voltage-gated Ca2+ channels.

Angiotensin II↗