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Biomedical subjects

L Shen

Publications and source records attributed to L Shen.

At least 217 records · Page 12Linked to original sources
PubMed1995

Location of the catalytic site for phosphoenolpyruvate formation within the primary structure of Clostridium symbiosum pyruvate phosphate dikinase. 1. Identification of an essential cysteine by chemical modification with [1-14C]bromopyruvate and site-directed mutagenesis.

Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (Pi), and pyruvate with adenosine 5'-monophosphate (AMP), pyrophosphate (PPi), and phosphoenolpyruvate (PEP). The reaction takes place according to the following steps: (1) E+ATP+P(i)<-->E-PP.AMP.P(i), (2) E-PP.AMP.P(i)<-->E-P+AMP+PP(i), and (3) E-P+pyruvate<-->E+PEP, where E represents free enzyme; E-PP, pyrophosphorylenzyme; and E-P, phosphorylenzyme. Steps 1 and 2 comprise the nucleotide partial reaction, and step 3 comprises the pyruvate partial reaction. The present studies were carried out to locate amino acid residues within the primary structure of Clostridium symbiosum PPDK participating in the catalysis of the pyruvate partial reaction. The enzyme was treated with the affinity label [1-14C]bromopyruvate, reduced with NaBH4, proteolyzed with trypsin, and chromatographed on an HPLC column. The radiolabeled tryptic peptide isolate was sequenced to reveal Cys 831 as the site of alkylation. Using PCR techniques Cys 831 was replaced by Ala, and the C831A PPDK mutant formed was then subjected to kinetic analysis. Rapid quench studies of single turnover reactions on the enzyme showed that the mutant is as efficient as wild-type PPDK in catalyzing the nucleotide partial reaction while it is unable to catalyze the pyruvate partial reaction. These results were interpreted as evidence for a role of Cys 831 in pyruvate/PEP binding and/or catalysis.

Affinity Labels↗
PubMed1995

Expression and functional characterization of chimeras between human and bovine vitamin-K-dependent protein-S-defining modules important for the species specificity of the activated protein C cofactor activity.

Vitamin-K-dependent protein S is an anticoagulant plasma protein functioning as a cofactor to activated protein C (APC) in the degradation of factors Va and VIIIa. The APC-cofactor function of protein S is species specific, as human protein S potentiates the anticoagulant activity of human but not that of bovine APC, whereas bovine protein S is a cofactor to APC from both species. To elucidate which modules in protein S determine the species specificity, in vitro mutagenesis was used to construct six recombinant chimeric molecules between human and bovine protein S. Wild-type human and bovine protein S and the chimeras were expressed in 293 cells and the recombinant proteins purified by monoclonal antibody affinity chromatography. The recombinant proteins were found to be post-translationally modified, they bound C4b-binding protein and were functionally active as cofactors to APC. Chimeras having both the thrombin-sensitive region (TSR) and the first epidermal-growth-factor-(EGF)-like module of bovine origin expressed APC-cofactor activity similar to that of bovine protein S. Those chimeras, in which TSR or EGF1 derived from different species, manifested APC-cofactor activity similar to that of human protein S, i.e. they did not express cofactor activity to bovine APC. These data indicate that sequence differences in the TSR and EGF1 of human and bovine protein S cause the species specificity of the APC-cofactor activity. The data support the concept that these two modules of protein S interact with APC on the surface of negatively charged phospholipids.

Amino Acid Sequence↗
PubMed1995

Characterization of a new four-chain coiled-coil: influence of chain length on stability.

Limited information is available on inherent stabilities of four-chain-coils. We have developed a model system to study this folding motif using synthetic peptides derived from sequences contained in the tetramerization domain of Lac repressor. These peptides are tetrameric as judged by both gel filtration and sedimentation equilibrium and the tetramers are fully helical as determined by CD. The four-chain coiled-coils are well folded as judged by the cooperativity of thermal unfolding and by the extent of dispersion in aliphatic chemical shifts seen in NMR spectra. In addition, we measured the chain length dependence of this four-chain coiled-coil. To this end, we developed a general procedure for nonlinear curve fitting of denaturation data in oligomeric systems. The dissociation constants for bundles that contain alpha-helical chains 21, 28, and 35 amino acids in length are 3.1 x 10(-12), 6.7 x 10(-23), and 1.0 x 10(-38) M3, respectively. This corresponds to tetramer stabilities (in terms of the peptide monomer concentration) of 180 microM, 51 nM, and 280 fM, respectively. Finally, we discuss the rules governing coiled-coil formation in light of the work presented here.

Amino Acid Sequence↗
PubMed1995

Kinematic analysis of cat hindlimb stepping.

1. The purpose of this study is to analyze the kinematics of stepping from the point of view of limb control by attempting to dissociate the various neuronal and mechanical factors that shape actual movements observed experimentally. The cat hindlimb, like the primate arm, is a multijointed mechanical system with redundant degrees of freedom. We contend that a number of issues studied in the context of arm movement control, such as end point control and trajectory planning, may also be applicable to limb movement in locomotion. 2. We recorded and analyzed the kinematics of cat hindlimb movement in unrestrained over-ground locomotion at various speeds and with a variety of surface conditions. We found we could represent limb movement in the step cycle simply and concisely by the trajectories of the limb segment orientation angles. Each trajectory conformed to a monophasic sawtoothlike waveform, and the relative timing between segments was largely invariant in the range of movements studied. This contrasts with the representation using relative joint angles, as in Philippson's scheme, which exhibits a monophasic waveform at the hip but biphasic waveforms at the knee and ankle. 3. To investigate how whole limb kinematics, i.e., changes in the length and orientation of the whole limb, relates to that of the limb segments, we reconstructed limb movement from segment trajectories, assuming as a first approximation that they were indeed sawtooth wave forms. The result strongly suggested that the relative timing of segment movements played an important role in regulating limb length during the swing phase of the step cycle. A strong correlation between segment relative timing and changes in limb length was also observed experimentally. 4. A comparison between reconstructed and actual limb movement revealed two major differences. In contrast with the actual movement, which exhibited at least two extension phases and limb shortening during the stance phase, the reconstructed movement had only a single extension phase and no limb shortening. The discrepancies were fully accountable, however, by the limb-ground interaction in stance, indicating that the features present in the actual movement resulted from the limb-ground interaction rather than from any elaborated control by the nervous system. 5. A second difference between reconstructed and actual movements was evident in an apparent jerkiness of the former and a difference in the hindpaw paths during the swing phase. These differences could be accounted for by including the consequences of limb inertia and finite muscle power, namely a gradual rather than instant change in velocity. Using a bell-shaped velocity profile for the segment movements, we were able to accurately reconstruct limb kinematics during the swing phase. 6. We conclude from this analysis that the overall features of limb kinematics in stepping may be controlled by regulating a small set of parameters related to the orientation angles of the limb segments. Specifically, the position of the endpoint, the hindpaw in this case, may be determined by the relative timing and amplitudes of segment trajectories. The point-by-point details of the observed limb kinematics may be largely attributed to limb mechanics and the interaction of the limb with its environment. Thus the neural control may be simpler than the kinematics suggests.

Animals↗
PubMed1995

The gene encoding vitamin K-dependent anticoagulant protein S is expressed in multiple rabbit organs as demonstrated by northern blotting, in situ hybridization, and immunohistochemistry.

Vitamin K-dependent protein S is an anticoagulant plasma protein that functions as a co-factor to activated protein C in the degradation of coagulation factors Va and VIIIa. We investigated the tissue/cellular distribution of protein S synthesis by Northern blotting, in situ hybridization, and immunohistochemistry. Northern blotting together with in situ hybridization, using specific oligodeoxynucleotide probes, demonstrated protein S mRNA in liver, lung, testis, epididymis, ovary, uterus, and brain. In the reproductive system, protein S mRNA was present in the cytoplasm of Leydig cells, interstitial cells of the ovary, epithelial cells of the epididymis, and in the endometrium, including endometrial mucous glandular membrane in the myometrium. Bronchial epithelial cells and alveolar macrophages were positive in the respiratory system. In the central nervous system, pyramidal neurons in the cerebral cortex and in the hippocampal region, and dentate fascia neurons gave strongly positive signals. Immunohistochemistry with monoclonal antibodies yielded a staining pattern that correlated well with results of in situ hybridization. In conclusion, results from Northern blotting, in situ hybridization, and immunohistochemistry suggested that rabbit protein S is expressed in several extrahepatic tissues. The presence of protein S transcripts in these fully differentiated cells suggests a cell type-specific gene expression which may be related to local anticoagulation or to other as yet unknown protein S functions.

Animals↗
PubMed1995

The gene encoding vitamin K-dependent anticoagulant protein C is expressed in human male reproductive tissues.

Protein C is a vitamin K-dependent protein circulating in plasma as a zymogen to an anticoagulant serine protease. After its activation, protein C cleaves and inactivates coagulation factors Va and VIIIa. Human protein C is synthesized in liver and undergoes extensive post-translational modification during its synthesis. Recently, the protein C inhibitor was demonstrated to be synthesized in several organs of the human male reproductive tract. Moreover, vitamin K-dependent protein S, which functions as a co-factor to activated protein C, was found to be synthesized in the Leydig cells of human testis. The aim of this study was to elucidate whether the protein C gene is also expressed in the male reproductive system. Specific immunostaining of protein C was found in Leydig cells of human testis, in the excretory epithelium of epididymis, and in some epithelial glands of the prostate, whereas no immunostaining was detected in seminal vesicles. Northern blotting and non-radioactive in situ hybridization demonstrated protein C mRNA in Leydig cells, in the excretory epithelium of epididymis, and in some of the epithelial glands of the prostate. The mRNA was distributed perinuclearly and the localization was in accordance with the specific immunostaining for protein C. The epithelium of epididymis was also found to contain both protein S mRNA and immunoreactivity. The demonstration of both protein C and protein S immunoreactivities, as well as their mRNAs, in male reproductive tissues suggests as yet unknown local functions for these proteins.

Aged↗
PubMed1995

Postmenopausal squamous atypia: a spectrum including "pseudo-koilocytosis".

In addition to typical atrophy, the postmenopausal cervix may exhibit a spectrum of epithelial and cellular alterations, including prominent perinuclear halos, nuclear hyperchromasia, variation in nuclear size, and multinucleation. It has not been determined whether such changes, termed postmenopausal squamous atypia (PSA), represent age-related epithelial disturbances or human papillomavirus (HPV)-related low-grade squamous intraepithelial lesions (condyloma). We surveyed 30 cervical biopsies from 26 women over the age of 50 that contained cytoplasmic halos and a spectrum of nuclear alterations, either alone or in association with atrophy. Twenty-three exhibited epithelium with 2- to 3-fold nuclear enlargement, and 18 had moderate or marked nuclear staining intensity. Eleven had a maximum of one or more multinucleated cells in a high-power field. Despite the nuclear alterations, none of the biopsies were positive for HPV by PCR analysis. This is in contrast to 104 of 141 low- and high-grade squamous intraepithelial lesions from a wide age range of women analyzed in the same manner (P = .000006). Features distinguishing PSA from HPV-associated low-grade squamous intraepithelial lesions (condyloma) included less variation in nuclear size and staining intensity, more finely and evenly distributed nuclear chromatin, and greater uniformity of perinuclear halos in PSA. In menopausal or postmenopausal women, PSA should be excluded when considering the diagnosis of a low-grade squamous intraepithelial lesion, specifically if the diagnosis rests on the interpretation of koilocytotic atypia.

Aged↗
PubMed1995

Mastoparan increases the intracellular free calcium concentration in two insulin-secreting cell lines by inhibition of ATP-sensitive potassium channels.

The mechanisms underlying mastoparan-induced elevation of the intracellular free calcium concentration ([Ca2+]i) were investigated in the insulin-secreting cell lines RINm5F and HIT. In both cell types, micromolar concentrations of mastoparan induced a prompt increase of [Ca2+]i, measured as an increase in fura-2 fluorescence. This response was dependent on extracellular calcium entry and was suppressed by organic calcium channel blockers; the increase of [Ca2+]i caused by high glucose concentrations or tolbutamide was not enhanced by mastoparan. These data indicate the involvement of voltage-dependent calcium channels and suggest that depolarization, rather than a direct effect on the channels, mediates the response to mastoparan. This proposition was supported by the observation that whole-cell calcium currents measured using the nystatin-permeabilized patch technique were not affected by mastoparan. Mastoparan-induced depolarization was observed using the potentiometric indicator bis-oxonol, and it was shown not to be additive with the depolarization induced by high glucose concentrations or tolbutamide. The mechanism underlying mastoparan-induced depolarization was identified in single-channel patch-clamp experiments, where it was shown that mastoparan caused closure of ATP-sensitive potassium channels [K(ATP) channels] in cell-attached and excised membrane patches. Responsiveness to mastoparan in excised patches demonstrated the membrane-delimited character of K(ATP) channel inhibition. The observation that the response persisted in the absence of exogenous GTP and in the presence of 250 microM GDP or guanosine-5'-O-(2-thio)diphosphate suggested that this effect is not mediated via enhancement of G protein activity. Partial suppression of channel activity by mastoparan did not prevent the action of tolbutamide, which fully suppressed the remaining activity in excised patches. In summary, the increase of [Ca2+]i in the insulin-secreting tumor cell lines RINm5F and HIT in response to mastoparan is mediated via G protein-independent suppression of K(ATP) channel activity, cell depolarization, and activation of voltage-dependent calcium channels.

Adenosine Triphosphate↗
PubMed1995

[Detecting human papillomavirus DNA of condylomata and pseudo-condylomata by in situ hybridization and polymerase chain reaction in paraffin-embedded specimens].

One hundred and thirty seven cases of paraffin-embedded tissue specimens biopsied each from condylomata and pseudo-condylomata of vulva and vagina were examined. Type 6b 11 human papillomavirus (HPV) DNA was detected in condylomata by in situ hybridization using digoxigeninlabelled probes and polymerase chain reaction (PCR). The positive rate was 82.5%, 83.9% respectively. It was negative in the 137 cases of pseudo-condylomata by in situ hybridization, but positive in 3 cases by PCR. For a correct diagnosis, the necessity of combining the data of clinical finding, microscopic examination and in situ hybridization or PCR is emphasized. Our results showed that pseudo-condylomata represent dysplastic appearances of vulval and vaginal mucosa, which become more marked during pregnancy and fungal or trichomonal infections, and could rather easily be misdiagnosed as condylomata.

Condylomata Acuminata↗
PubMed1995

[Analysis of penbutolol and its metabolites in human body fluid by gas chromatography--mass spectrometry].

A method for the detection and identification of penbutolol and its metabolites in human body fluid by using gas chromatography-mass spectrometry was established. The urine was extracted with diethyl ether-isopropanol and the plasma was extracted with Sep-Pak column. After TMS derivatization the sample was analyzed with GC-MSD. Six metabolites of penbutolol were found in urine sample. The recovery was 90.83% for urine and 85.88% for plasma. The detection limit of penbutolol was 5 pg.

Adrenergic beta-Antagonists↗
PubMed1995

[Study on the perinatal infection caused by Chlamydia trachomatis].

OBJECTIVE: Study on the frequency and outcome of chlamydia trachomatis (CT) infection of the pregnant women and their babies. METHODS: Two hundred and seventy eight pregnant women and their 79 infants were studied with McCoy cell culture, polymerase chain reaction (PCR) and DNA sequence analysis. Isolation of CT was performed in McCoy cell culture, and iodine-stained glycogen-positive inclusions were considered to be positive. CT DNAs were extracted by modified NaI method. Two sets of primers derived from CT common plasmid and major outer membrane protein genes were used in the PCR. Dideoxy-mediated chain-termination method was used for DNA sequence analysis. RESULTS: 10.8% (30/278) of the cervical cultures were positive for CT, while the positive rate by PCR was 14.0% (39/278). The vertical transmission rate of CT was 55.0% (11/22). The sequences of DNA amplified from a maternal-infant pair were matched perfectly. The incidence of puerperal endometritis in CT positive women was 28.6%, much higher than 8.1% in those CT negative mothers. Neonatal conjunctivitis and pneumonia in group of CT positive mothers were 45.0% and 30.0%, respectively, much higher than 18.3% and 8.3%, respectively, in that of CT negative mothers (P < 0.05). CONCLUSION: Perinatal infection caused by CT is very common in China.

Chlamydia Infections↗
PubMed1994

The repertoire of cytotoxic T lymphocytes in the recognition of mutant simian immunodeficiency virus variants.

The importance of the repertoire of a primed, AIDS virus-specific population of CTL in the recognition of emerging mutant viruses was assessed in simian immunodeficiency virus (SIV)-infected rhesus monkeys. These studies were done by using the well-characterized CTL recognition of the SIVmac Gag peptide 11C (p11C) epitope in rhesus monkeys expressing the MHC class I molecule Mamu-A*01. Lysis of peptide-pulsed targets by bulk PBL effector cells from SIVmac-infected, Mamu-A*01+ monkeys was significantly decreased by mutations at residues 2, 3, and 5 of the nine-amino-acid Gag p11C epitope. However, effector cell lysis of targets pulsed with p11C containing substitutions at residues 3 or 5 was substantially increased by in vitro incubation of the monkeys' PBL with these mutant peptides. This suggested that expandable populations of cells exist in the primed CTL of infected monkeys capable of recognizing mutant peptide sequences. In fact, without in vitro exposure of PBL to mutant peptides, SIVmac Gag-specific CTL could be cloned from PBL of infected monkeys that lysed targets pulsed with mutant peptides. These observations suggest that clones of effector cells capable of recognizing many viral mutants may be able to expand in vivo to control the spread of emerging variant AIDS viruses.

Amino Acid Sequence↗
PubMed1994

Vitamin K-dependent protein S in Leydig cells of human testis.

Protein S is an anticoagulant plasma protein, functioning as a cofactor to activated protein C in the regulation of blood coagulation. In addition, protein S forms a complex with the complement regulatory protein, C4b-binding protein. Protein S is unique among the vitamin K-dependent proteins in being structurally similar to androgen binding proteins. Protein S immunoreactivity was demonstrated in Leydig cells of human testis. In Northern blotting experiments, the presence of protein S mRNA in human testis tissue could be shown. In situ hybridization experiments localized protein S mRNA to the Leydig cells, demonstrating transcription of the protein S gene in these cells. Five protein S clones were isolated from a human testis cDNA library, partially sequenced and characterized by restriction enzyme mapping. Three unique clones contained information for the entire coding sequence and approximately two-thirds of the 5' and 3' non-coding sequences. The results indicate the nucleotide sequences of testis and liver protein S mRNA to be identical. No binding of androgens to protein S could be demonstrated. In conclusion, we demonstrate the presence of protein S immunoreactivity as well as protein S mRNA in the Leydig cells of human testis. These results suggest local synthesis of protein S in Leydig cells of human testis which may be functionally important for local anticoagulation.

Base Sequence↗
PubMed1994

An acutely lethal simian immunodeficiency virus stimulates expansion of V beta 7- and V beta 14-expressing T lymphocytes.

SIVsmmPBj14, a variant simian immunodeficiency virus isolated from a pig-tailed macaque, stimulates the proliferation of macaque T lymphocytes in vitro and induces an acutely lethal disease in macaques characterized, in part, by lymphadenopathy and splenomegaly. To determine whether SIVsmmPBj14 exhibits superantigen-like activity, in vitro and in vivo studies of T-cell receptor V beta repertoire were undertaken using PCR-based quantitative methods. Whereas in vitro phytohemagglutinin stimulation of macaque peripheral blood lymphocytes did not cause a perturbation of T-cell receptor V beta repertoire, SIVsmmPBj14 stimulated the expansion of both CD4+ and CD8+ T-lymphocyte subpopulations expressing the V beta 7 and V beta 14 gene families. Such V beta 7 and V beta 14 expansions could be confirmed by a multiple RNase protection assay. Furthermore, the expansion of the same lymphocyte subpopulations was also detected in peripheral blood lymphocytes and lymph node cells of virus-infected macaques. These observations suggest that SIVsmmPBj14-mediated V beta expansion may contribute to the induction of an acutely lethal disease in macaques.

Acute Disease↗
PubMed1994

Factor V and protein S as synergistic cofactors to activated protein C in degradation of factor VIIIa.

Inherited resistance to activated protein C (APC) is a recently identified major cause of thrombosis. It is associated with a mutation in the factor V gene affecting one of the cleavage sites for APC. APC resistance was recently found to be corrected by factor V, suggesting that factor V may have anticoagulant properties as a cofactor to APC. To elucidate this further, we have studied the effect of factor V and protein S, which is a known cofactor to APC, on APC-mediated degradation of factor VIIIa in a purified system. The APC-mediated degradation of factor VIIIa was monitored by a factor X activation reaction using purified factor IXa, phospholipid, and calcium. In the presence of both factor V and protein S, APC was found to inhibit factor VIIIa activity efficiently. APC alone or together with factor V was ineffective, whereas APC in combination with protein S was less efficient than when factor V was also included in the reaction. Two monoclonal antibodies, one against protein S and the other directed toward factor V, were found to inhibit the APC cofactor activity of the factor V-protein S mixture. Factor Va did not express APC cofactor activity, and addition of excess factor Va over factor V did not inhibit the APC cofactor function of a factor V-protein S mixture. In conclusion, the results suggest that factor V and protein S work in synergy as phospholipid-bound cofactors to APC.

Enzyme Activation↗
PubMed1994

Molecular cloning and expression of early T cell costimulatory molecule-1 and its characterization as B7-2 molecule.

The interaction of T cell CD28/CTLA-4 receptors with B7-1 activation Ag on APC represents an important costimulatory pathway in T cell activation. However, it is now evident that this costimulatory pathway is neither unique nor universal for the activation of T cells. Our previous study indicated that a 60-kDa membrane protein, recognized by mAb 2D10, was expressed before B7 by activated murine B cells. This molecule was critically involved in activation of T cells in response to auto- and alloantigens. In the present study, we report on the isolation of a cDNA for this early T cell costimulatory molecule (ETC-1). ETC-1, like B7-1, is a member of the Ig supergene family and is composed of 303 amino acids. Nucleic acid sequence comparison indicated that ETC-1 is identical to the B7-2 molecule. When expressed in Chinese hamster ovary cells, ETC-1 showed profound T cell costimulatory activity as demonstrated by its ability to enhance CD4 T cell proliferation in response to Con A or anti-CD3 stimulation. Furthermore, ETC-1 also bound to both CD28-Ig and CTLA4-Ig fusion proteins. These results strongly support the notion that the interaction of ETC-1/B7-2 with CD28 or CTLA-4 receptors represents an alternative T cell costimulatory pathway.

Abatacept↗
PubMed1994

Lipopolysaccharide and cytokine augmentation of human monocyte IgA receptor expression and function.

Receptors for IgA (Fc alpha R) are found on phagocytic cells in the peripheral blood and tissues associated with mucosal areas where IgA Abs constitute a major line of defense. Because Fc alpha R are capable of triggering protective functions of monocytes and neutrophils, such as phagocytosis and the oxidative burst, they may be important in amplifying the antimicrobial effects of IgA. Various cytokines play a role in regulating function and FcR expression of monocytes, macrophages, and neutrophils. The present studies examine the modulation of monocyte Fc alpha R by LPS and cytokines. LPS strongly up-regulated monocyte Fc alpha R expression. TNF and IL-1, produced in response to LPS, promoted Fc alpha R increase, as did GM-CSF; whereas IFN-gamma down-regulated Fc alpha R. Increased receptor expression was accompanied by augmented IgA-mediated phagocytosis. An increase in Fc alpha R-specific mRNA was detected in monocytes treated with TNF, IL-1, GM-CSF, and LPS; whereas message was reduced in cells treated with IFN-gamma. Monocyte-derived macrophages and cells of the Monomac 6 monocyte-like line expressed greater numbers of Fc alpha R than monocytes but were less responsive to LPS and TNF. Cell lines THP-1 and U937, which expressed similar or lower levels of Fc alpha R than monocytes, displayed an increase in Fc alpha R in response to LPS and, to various degrees, to TNF, IL-1, and GM-CSF. These results indicate that Fc alpha R on monocytes are modulated by endotoxin and an array of cytokines distinct from those that regulate expression of FcR for IgG.

Cell Line↗